Subtilase variants and compositions comprising same

ABSTRACT

The invention relates to subtilase variants and detergent compositions comprising the variants, as well as methods of producing the variants and methods for stabilizing a subtilase variant.

REFERENCE TO A SEQUENCE LISTING

This application contains a sequence listing in computer readable form,which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to subtilase variants, compositionscomprising the variants, polynucleotides encoding the variants, methodsof producing the variants, and methods of using the variants.

BACKGROUND OF THE INVENTION

Subtilisins are serine proteases from the family S8, in particular fromthe subfamily S8A, as defined by the MEROPS database(https://www.ebi.ac.uk/merops/index.shtml). In subfamily S8A the keyactive site residues Asp, His and Ser are typically found in motifs thatdiffer from those of the S8B subfamily. Subtilisin BPN′ (also knownunder the acronym BASBPN) from Bacillus amyloliquefaciens has the MEROPSnumbers S08.034 and is a member of the S8A subfamily.

In the detergent industry, enzymes have for many decades beenimplemented in washing formulations. Enzymes used in such formulationscomprise proteases, lipases, amylases, cellulases, mannosidases as wellas other enzymes or mixtures thereof. Commercially, the most importantenzymes are proteases.

An increasing number of commercially used proteases for e.g. laundry anddishwashing detergents are protein engineered variants of naturallyoccurring wild type proteases, Further, other subtilase variants havebeen described in the art with alterations relative to a parentsubtilase resulting in improvements such as better wash performance,thermal stability, storage stability or catalytic activity.

However, various factors make further improvement of proteasesadvantageous. For example, washing conditions such as temperature and pHtend to change over time, and are also different in different countriesor regions of the world, and many stains are still difficult tocompletely remove under conventional washing conditions. Anotherchallenge in detergent compositions is enzyme stability, since thechemical components of these compositions as well as conditions of pH,temperature and humidity often tend to inactivate enzymes. Further,in-wash conditions can also result in inactivation of the enzymes (dueto e.g. pH, temperature or chelation instability), resulting in loss ofwash performance during the wash cycle. Thus, despite the intensiveresearch in protease development there remains a need for new andimproved proteases that have improved stability, for example improvedstorage stability, e.g. in a detergent composition, and which at thesame time have similar or improved wash performance compared to theparent subtilase.

The present invention addresses these challenges by providing subtilasevariants with improved stability.

SUMMARY OF THE INVENTION

The present invention relates to subtilase variants, comprising analteration at two, three or more positions, e.g. four or more positions,corresponding to positions 9, 63, 76, 88, 104, 107, 128, 131, 159, 204,206, 209, 212, 215, 216, 261 and 262 of the polypeptide of SEQ ID NO: 1,wherein the variants have subtilase activity.

The present invention also relates to compositions comprising thevariants, in particular detergent compositions, polynucleotides encodingthe variants; nucleic acid constructs, vectors, and host cellscomprising the polynucleotides; and methods of producing the variants.

The present invention further relates to methods for stabilizing asubtilase variant and for producing a subtilase variant.

Definitions

Subtilase/protease: The terms “subtilase” and “protease” may be usedinterchangeably herein and refer to an enzyme that hydrolyses peptidebonds in proteins. This includes any enzyme belonging to the EC 3.4enzyme group (including each of the thirteen subclasses thereof), and inparticular endopeptidases (EC 3.4.21). The EC number refers to EnzymeNomenclature 1992 from NC-IUBMB, Academic Press, San Diego, Calif.,including supplements 1-5 published in Eur. J. Biochem. 1994, 223, 1-5;Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur.J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650;respectively.

Protease activity: The term “protease activity” means a proteolyticactivity (EC 3.4), in particular endopeptidase activity (EC 3.4.21).There are several protease activity types, the three main activity typesbeing: trypsin-like, where there is cleavage of amide substratesfollowing Arg or Lys at P1, chymotrypsin-like, where cleavage occursfollowing one of the hydrophobic amino acids at P1, and elastase-likewith cleavage following an Ala at P1. For purposes of the presentinvention, protease activity is determined according to the proceduredescribed in “Materials and Methods” below. The subtilisin variants ofthe present invention preferably have at least 50%, e.g. at least 60%,at least 70%, at least 80%, at least 90%, at least 95% or at least 100%of the protease activity of the polypeptide of SEQ ID NO: 1.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial primary RNAtranscript is a precursor to mRNA that is processed through a series ofsteps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide whichdirectly specifies the amino acid sequence of a variant. The boundariesof the coding sequence are generally determined by an open readingframe, which begins with a start codon such as ATG, GTG or TTG and endswith a stop codon such as TAA, TAG, or TGA. The coding sequence may be agenomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Fragment: The term “fragment” means a polypeptide having one or moreamino acids absent from the amino and/or carboxyl terminus of a maturepolypeptide; wherein the fragment has subtilase activity. Such afragment preferably contains at least 85%, at least 90% or at least 95%of the number of amino acids in SEQ ID NO: 1.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parentprotease or the protease with SEQ ID NO: 1. Such improved propertiesinclude, but are not limited to, catalytic efficiency, catalytic rate,chemical stability, oxidation stability, pH activity, pH stability,specific activity, stability under storage conditions, substratebinding, substrate cleavage, substrate specificity, substrate stability,surface properties, thermal activity and thermostability. In a preferredaspect of the present invention, the improved property is improvedstability, in particular improved storage stability in a detergentformulation, where the storage stability may be determined based onculture supernatants or on purified proteases, e.g. as described in theexamples herein.

Isolated: The term “isolated” means a substance in a form or environmentwhich does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving subtilase activity.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent or parent subtilase/protease: The term “parent” or “parentsubtilase” or “parent protease” means any polypeptide with subtilaseactivity to which an alteration is made to produce the enzyme variantsof the present invention. The parent may be a naturally occurring(wild-type) polypeptide or a variant thereof of a wild-type polypeptide.In a particular embodiment, the parent is a protease with at least 75%identity, such as at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98% or at least 99% identity with SEQ IDNO: 1. Alternatively, the parent may have 100% identity to SEQ ID NO: 1.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Variant: The term “variant” means a polypeptide having subtilaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more positions. A substitution meansreplacement of the amino acid occupying a position with a differentamino acid; a deletion means removal of the amino acid occupying aposition; and an insertion means adding an amino acid adjacent to andimmediately following the amino acid occupying a position.

Wild-type subtilase: The term “wild-type” subtilase means a subtilaseexpressed by a naturally occurring microorganism, such as a bacterium,yeast, or filamentous fungus found in nature. An example of a wild-typesubtilase is subtilisin BPN′, i.e., amino acids 1 to 275 of SEQ ID NO:1.

Conventions for Designation of Variants

For purposes of the present invention, the polypeptide with SEQ ID NO: 1is used to determine the corresponding amino acid residue in anothersubtilase. The amino acid sequence of another subtilase is aligned withthe polypeptide disclosed in SEQ ID NO: 1, and based on the alignment,the amino acid position number corresponding to any amino acid residuein the polypeptide of SEQ ID NO: 1 is determined using theNeedleman-Wunsch algorithm as described above under “Sequence identity”and with the parameters described above.

Identification of the corresponding amino acid residue in anothersubtilase can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537:_39-64; Katoh and Toh, 2010,Bioinformatics 26:_1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed. Theterms “alteration” or “mutation” may be used interchangeably herein torefer to substitutions, insertions and deletions.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of a threonine at position 220 withalanine is designated as “Thr220Ala” or “T220A”. Multiple substitutionsmay be separated by addition marks (“+”), e.g., “Thr220Ala+Gly229Val” or“T220A+G229V”, representing substitutions at positions 220 and 229 ofthreonine (T) with alanine (A) and glycine (G) with valine (V),respectively. Multiple substitutions may alternatively be listed withindividual mutations separated by a space or a comma. Alternativesubstitutions in a particular position may be indicated with a slash(“/”). For example, substitution of threonine in position 220 witheither alanine, valine or leucine many be designated “T220A/V/L”.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofthreonine at position 220 is designated as “Thr220*” or “T220*”.Multiple deletions may be separated by addition marks (“+”), e.g.,“Thr220*+Gly229*” or “T220*+G229*”, or alternatively may be separated bya space or comma.

Insertions. For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, inserted aminoacid. Accordingly, the insertion of lysine after threonine at position220 is designated “Thr220ThrLys” or “T220TK”. An insertion of multipleamino acids is designated [Original amino acid, position, original aminoacid, inserted amino acid #1, inserted amino acid #2; etc.]. Forexample, the insertion of lysine and alanine after threonine at position220 is indicated as “Thr220ThrLysAla” or “T220TKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 220 220 220a 220b T T - K - A

Multiple alterations. Variants comprising multiple alterations areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine atpositions 170 and 195 with tyrosine and glutamic acid, respectively.Multiple alterations may alternatively be listed with individualmutations separated by a space or a comma.

Different alterations. Where different alterations can be introduced ata position, the different alterations may be separated by a comma, e.g.,“Arg170Tyr,Glu” represents a substitution of arginine at position 170with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala”designates the following variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and“Tyr167Ala+Arg170Ala”.

Different alterations in a position may also be indicated with a slash(“/”), for example “T220A/V/L” as explained above. Alternatively,different alterations may be indicated using brackets, e.g., Arg170[Tyr,Gly] or in one-letter code R170 [Y,G].

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to subtilase variants comprising mutationsat three or more positions, e.g. four or more positions, selected from9, 63, 76, 88, 104, 107, 128, 131, 159, 204, 206, 209, 212, 215, 216,261 and 262, wherein positions are numbered according to SEQ ID NO: 1,and wherein the variant has protease activity and a sequence identity toSEQ ID NO: 1 of at least 80% and less than 100%.

Variants

In one embodiment, the invention provides a subtilase variant comprisingmutations at three or more positions, e.g. four or more positions,selected from:

-   -   (i) 209 in combination with two or more mutations at positions        selected from 63, 215 and 217;    -   (ii) 104 and 128 in combination with at least one mutation at a        position selected from 9, 63, 76, 107, 131, 159, 204, 206, 212,        215, 216 and 217;    -   (iii) 9 in combination with at least two mutations at positions        selected from 76, 204 and 212;    -   (iv) 76 in combination with at least two mutations at positions        selected from 9, 88, 159, 204, 206, 212, 216, 261 and 262;    -   (v) 206 in combination with at least two mutations at positions        selected from 9, 76, 159, 204, 209, 212, 216, 217, 261 and 262;        and    -   (vi) 204, 212 and 216, preferably in combination with at least        one mutation at a position selected from 9, 159 and 206;    -   wherein positions are numbered according to SEQ ID NO: 1, and        wherein the variant has protease activity and a sequence        identity to SEQ ID NO: 1 of at least 80%, for example at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, or at least        98%, but less than 100%.

In some embodiments, the mutations in positions 9, 63, 76, 88, 104, 107,128, 131, 159, 204, 206, 209, 212, 215, 216, 261 and 262 are selectedfrom the following:

-   -   the mutation in position 9 is S9E or S9D, preferably S9E;    -   the mutation in position 63 is S63G or S63A, preferably S63G;    -   the mutation in position 76 is N76D or N76E, preferably N76D;    -   the mutation in position 88 is A88V, A881, A88L or A88M,        preferably A88V;    -   the mutation in position 104 is Y104V, Y104I, Y104L or Y104M,        preferably Y104V;    -   the mutation in position 107 is I107L, I107V, I107M, preferably        I107L;    -   the mutation in position 128 is G128S, G128A or G128T,        preferably G128S;    -   the mutation in position 131 is G131* or G131P;    -   the mutation in position 159 is S159E or S159D, preferably        S159E;    -   the mutation in position 204 is S204D or S204E, preferably        S204D;    -   the mutation in position 206 is Q206L, Q206I, Q206V or Q206M,        preferably Q206L;    -   the mutation in position 209 is L209W;    -   the mutation in position 212 is N212G, N212A or N212S,        preferably N212G;    -   the mutation in position 215 is G215A;    -   the mutation in position 216 is A216V, A216I, A216L or A216M,        preferably A216V;    -   the mutation in position 217 is Y217L, Y217I, Y217V or Y217M,        preferably Y217L;    -   the mutation in position 261 is F261W, F261N or F261Y,        preferably F261W; and/or    -   the mutation in position 262 is Y262E or Y262D, preferably        Y262E.

In another embodiment, the invention provides a subtilase variantcomprising three or more mutations, e.g. four or more mutations,selected from:

-   -   (i) L209W in combination with two or more substitutions selected        from S63G/A, G215A and Y217L/I/V/M;    -   (ii) Y104V/I/L/M and G128S/A/T in combination with at least one        mutation selected from S9E/D, S63G/A, N76D/E, I107L/V/M, G131*,        G131P, S159E/D, S204D/E, Q206L/I/V/M, N212G/A/S, G215A,        A216V/I/L/M and Y217L/I/V/M;    -   (iii) S9E/D in combination with at least two substitutions        selected from N76D/E, S204D/E and N212G/A/S;    -   (iv) N76D/E in combination with at least two substitutions        selected from S9E/D, A88V/I/L/M, S159E/D, S204D/E, Q206L/I/V/M,        N212G/A/S, A216V/I/L/M, F261W/N/Y and Y262E/D;    -   (v) Q206L/I/V/M in combination with at least two substitutions        selected from S9E/D, N76D/E, S159E/D, S204D/E, L209W/N/Y,        N212G/A/S, A216V/I/L/M, Y217L/I/V/M, F261W/N/Y and Y262E/D; and    -   (vi) S204D/E, N212G/A/S and A216V/I/L/M, preferably in        combination with at least one substitution selected from S9E/D,        S159E/D and Q206L/I/V/M;    -   wherein positions are numbered according to SEQ ID NO: 1, and        wherein the variant has protease activity and a sequence        identity to SEQ ID NO: 1 of at least 80%, for example at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, or at least        98%, but less than 100%.

In a first aspect of the invention, the subtilase variant comprises orconsists of the substitution L209W and at least two substitutionsselected from the group consisting of S63G/A, G215A and Y217L/I/V/M,wherein positions are numbered according to SEQ ID NO: 1, and whereinthe variant has protease activity and a sequence identity to SEQ ID NO:1 of at least 80%, for example at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, or at least 98%, but less than 100%.

The substitution in position 63 is preferably S63G, the substitution inposition 215 is preferably G215A, and the substitution in position 217is preferably Y217L. In a preferred embodiment, the subtilase variant ofthis aspect comprises each of the substitutions S63G, L209W, G215A andY217L.

The subtilase variant of this aspect, comprising the substitution L209Wand at least two substitutions selected from the group consisting ofS63G/A, G215A and Y217L/I/V/M, wherein the substitution in position 63preferably is S63G, the substitution in position 215 preferably is G215Aand the substitution in position 217 preferably is Y217L, may furthercomprise at least one mutation, for example two, three or moremutations, selected from the group consisting of S9E/D, N76D/E,Y104V/I/L/M, G128S/A/T, G131*, G131P, S204D/E, Q206L/I/V/M, A216V/I/L/M,F261W/N/Y and Y262E/D.

In one embodiment, the subtilase variant comprises the substitutionsS63G/A, L209W, G215A and Y217L/I/V/M, preferably S63G, L209W, G215A andY217L, and further comprises a substitution in position 9 selected fromS9E and S9D, preferably S9E.

In another embodiment, the subtilase variant comprises the substitutionsS63G/A, L209W, G215A and Y217L/I/V/M, preferably S63G, L209W, G215A andY217L, and further comprises a substitution in position 76 selected fromN76D and N76E, preferably N76E.

In another embodiment, the subtilase variant comprises the substitutionsS63G/A, L209W, G215A and Y217L/I/V/M, preferably S63G, L209W, G215A andY217L, and further comprises a substitution in position 104 selectedfrom Y104V, Y104I, Y104L and Y104M, preferably Y104V.

In another embodiment, the subtilase variant comprises the substitutionsS63G/A, L209W, G215A and Y217L/I/V/M, preferably S63G, L209W, G215A andY217L, and further comprises a substitution in position 128 selectedfrom G128S, G128A and G128T, preferably G128S.

In another embodiment, the subtilase variant comprises the substitutionsS63G/A, L209W, G215A and Y217L/I/V/M, preferably S63G, L209W, G215A andY217L, and further comprises an alteration in position 131, wherein thealteration is selected from G131* and G131P.

In another embodiment, the subtilase variant comprises the substitutionsS63G/A, L209W, G215A and Y217L/I/V/M, preferably S63G, L209W, G215A andY217L, and further comprises a substitution in position 204 selectedfrom S204D and S204E, preferably S204D.

In another embodiment, the subtilase variant comprises the substitutionsS63G/A, L209W, G215A and Y217L/I/V/M, preferably S63G, L209W, G215A andY217L, and further comprises a substitution in position 206 selectedfrom Q206L, Q206I, Q206V and Q206M, preferably Q206L.

In another embodiment, the subtilase variant comprises the substitutionsS63G/A, L209W, G215A and Y217L/I/V/M, preferably S63G, L209W, G215A andY217L, and further comprises a substitution in position 216 selectedfrom A216V, A216I, A216L and A216M, preferably A216V.

In another embodiment, the subtilase variant comprises the substitutionsS63G/A, L209W, G215A and Y217L/I/V/M, preferably S63G, L209W, G215A andY217L, and further comprises a substitution in position 261 selectedfrom F261W, F261N and F261Y, preferably F261W.

In another embodiment, the subtilase variant comprises the substitutionsS63G/A, L209W, G215A and Y217L/I/V/M, preferably S63G, L209W, G215A andY217L, and further comprises a substitution in position 262 selectedfrom Y262E and Y262D, preferably Y262E.

In another embodiment, the subtilase variant comprises the substitutionS63G/A, L209W, G215A and Y217L/I/V/M, preferably S63G, L209W, G215A andY217L, and further comprises a substitution in position 206 selectedfrom selected from Q206L, Q206I, Q206V and Q206M, preferably Q206L, anda substitution in position 216 selected from A216V, A216I, A216L andA216M, preferably A216V.

In another embodiment, the subtilase variant comprises the substitutionS63G/A, L209W, G215A and Y217L/I/V/M, preferably S63G, L209W, G215A andY217L, and further comprises a substitution in position 261 selectedfrom F261W, F261N and F261Y, preferably F261W, and a substitution inposition 262 selected from Y262E and Y262D, preferably Y262E.

In another embodiment, the subtilase variant comprises the substitutionS63G/A, L209W, G215A and Y217L/I/V/M, preferably S63G, L209W, G215A andY217L, and further comprises a substitution in position 104 selectedfrom Y104V, Y104I, Y104L and Y104M, preferably Y104V, and a substitutionin position 128 selected from G128S, G128A and G128T, preferably G128S.

In another embodiment, the subtilase variant comprises the substitutionS63G/A, L209W, G215A and Y217L/I/V/M, preferably S63G, L209W, G215A andY217L, and further comprises a substitution in position 104 selectedfrom Y104V, Y104I, Y104L and Y104M, preferably Y104V, a substitution inposition 128 selected from G128S, G128A and G128T, preferably G128S, andan alteration in position 131, wherein the alteration is selected fromG131* and G131P.

In a particular embodiment of this aspect of the invention, thesubtilase variant comprises or consists of the set of mutations S63GL209W G215A Y217L.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations S9E S63G L209W G215A Y217L.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations S63G N76D L209W G215A Y217L.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations S63G S204D L209W G215A Y217L.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations S63G Q206L L209W G215A A216V Y217L.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations S63G Q206L L209W G215A Y217L.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations S63G L209W G215A Y217L F261W Y262E.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations S63G Y104V G128S G131* L209W G215AY217L.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations S63G Y104V G128S G131P L209W G215AY217L.

In a second aspect of the invention, the subtilase variant comprises orconsists of the substitutions Y104V/I/L/M+G128S/A/T, preferablyY104V+G128S, and (a) at least two mutations selected from the groupconsisting of S9E/D, N76D/E, I107L/V/M, G131*, G131P, S159E/D, S204D/E,Q206L/I/V/M, N212G/A/S and A216V/I/L/M, or (b) L209W and at least twosubstitutions selected from the group consisting of S63G/A, G215A andY217L/I/V/M, wherein positions are numbered according to SEQ ID NO: 1,and wherein the variant has protease activity and a sequence identity toSEQ ID NO: 1 of at least 80%, for example at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, or at least 98%, but less than 100%.

In one embodiment of this aspect of the invention, the subtilase variantcomprises the substitutions Y104V/I/L/M+G128S/A/T, preferablyY104V+G128S, and further comprises a substitution in position 9 selectedfrom S9E and S9D, preferably S9E.

In another embodiment, the subtilase variant comprises the substitutionsY104V/I/L/M+G128S/A/T, preferably Y104V+G128S, and further comprises asubstitution in position 76 selected from N76D and N76E, preferablyN76E.

In another embodiment, the subtilase variant comprises the substitutionsY104V/I/L/M+G128S/A/T, preferably Y104V+G128S, and further comprises asubstitution in position 107 selected from I107L, I107V and I107M,preferably I107L.

In another embodiment, the subtilase variant comprises the substitutionsY104V/I/L/M+G128S/A/T, preferably Y104V+G128S, and further comprises asubstitution in position 131 selected from G131* and G131P.

In another embodiment, the subtilase variant comprises the substitutionsY104V/I/L/M+G128S/A/T, preferably Y104V+G128S, and further comprises asubstitution in position 159 selected from S159E and S159D, preferably5159E.

In another embodiment, the subtilase variant comprises the substitutionsY104V/I/L/M+G128S/A/T, preferably Y104V+G128S, and further comprises asubstitution in position 204 selected from S204D and S204E, preferablyS204D.

In another embodiment, the subtilase variant comprises the substitutionsY104V/I/L/M+G128S/A/T, preferably Y104V+G128S, and further comprises asubstitution in position 206 selected from Q206L, Q206I, Q206V andQ206M, preferably Q206L.

In another embodiment, the subtilase variant comprises the substitutionsY104V/I/L/M+G128S/A/T, preferably Y104V+G128S, and further comprises asubstitution in position 212 selected from N212G, N212A and N212S,preferably N212G.

In another embodiment, the subtilase variant comprises the substitutionsY104V/I/L/M+G128S/A/T, preferably Y104V+G128S, and further comprises asubstitution in position 216 selected from A216V, A216I, A216L andA216M, preferably A216V.

In another embodiment, the subtilase variant comprises the substitutionsY104V/I/L/M+G128S/A/T, preferably Y104V+G128S, and further comprises asubstitution in position 76 selected from N76D and N76E, preferablyN76D, and a mutation in position 131 selected from G131* and G131P.Exemplary variants of this embodiment comprise or consist of thesubstitutions N76D+Y104V+G128S+G131* or N76D+Y104V+G128S+G131P.

In another embodiment, the subtilase variant comprises the substitutionsY104V/I/L/M+G128S/A/T, preferably Y104V+G128S, and further comprises asubstitution in position 76 selected from N76D and N76E, preferablyN76D, a mutation in position 131 selected from G131* and G131P, and asubstitution in position 212 selected from N212G, N212A and N212S,preferably N212G. Exemplary variants of this embodiment comprise orconsist of the substitutions N76D+Y104V+G128S+G131*+N212G orN76D+Y104V+G128S+G131P+N212G.

In another embodiment, the subtilase variant comprises the substitutionsY104V/I/L/M+G128S/A/T, preferably Y104V+G128S, and further comprises amutation in position 131 selected from G131* and G131P, and asubstitution in position 204 selected from S204D and S204E, preferablyS204D. Exemplary variants of this embodiment comprise or consist of thesubstitutions Y104V+G128S+G131*+Q204D or Y104V+G128S+G131P+Q204D.

In another embodiment, the subtilase variant comprises the substitutionsY104V/I/L/M+G128S/A/T, preferably Y104V+G128S, and further comprises amutation in position 131 selected from G131* and G131P, and asubstitution in position 206 selected from Q206L, Q206I, Q206V andQ206M, preferably Q206L. Exemplary variants of this embodiment compriseor consist of the substitutions Y104V+G128S+G131*+Q206L orY104V+G128S+G131P+Q206L.

In another embodiment, the subtilase variant comprises the substitutionsY104V/I/L/M+G128S/A/T, preferably Y104V+G128S, and further comprises amutation in position 131 selected from G131* and G131P, and asubstitution in position 212 selected from N212G, N212A and N212S,preferably N212G. Exemplary variants of this embodiment comprise orconsist of the substitutions Y104V+G128S+G131*+N212G orY104V+G128S+G131P+N212G.

In another embodiment, the subtilase variant comprises the substitutionsY104V/I/L/M+G128S/A/T, preferably Y104V+G128S, and further comprises amutation in position 131 selected from G131* and G131P, and asubstitution in position 216 selected from A216V, A216I, A216L andA216M, preferably A216V. Exemplary variants of this embodiment compriseor consist of the substitutions Y104V+G128S+G131*+A216V orY104V+G128S+G131P+A216V.

In another embodiment, the subtilase variant comprises the substitutionsY104V/I/L/M+G128S/A/T, preferably Y104V+G128S, and further comprises amutation in position 131 selected from G131* and G131P, and asubstitution in position 159 selected from S159E and S159D, preferablyS159E. Exemplary variants of this embodiment comprise or consist of thesubstitutions Y104V+G128S+G131*+S159E or Y104V+G128S+G131P+S159E.

In another embodiment, the subtilase variant comprises the substitutionsY104V/I/L/M+G128S/A/T, preferably Y104V+G128S, and further comprises asubstitution in position 9 selected from S9E and S9D, preferably S9E,and a mutation in position 131 selected from G131* and G131P. Exemplaryvariants of this embodiment comprise or consist of the substitutionsY104V+G128S+S9E+G131* or Y104V+G128S+S9E+G131P.

In a particular embodiment of this aspect of the invention, thesubtilase variant comprises or consists of the set of mutations N76DY104V G128S G131* N212G.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations N76D Y104V G128S G131P.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations N76D Y104V G128S G131*.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations Y104V I107L G128S G131* Q206L.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations N76D Y104V G128S G131P N212G.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations S63G Y104V G128S G131* L209W G215AY217L.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations Y104V G128S G131* Q206L A216V.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations S63G Y104V G128S G131P L209W G215AY217L.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations Y104V G128S G131* Q206L.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations Y104V G128S G131* S159E Q206L A216V.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations Y104V G128S G131* S204D N212G.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations Y104V G128S G131P S204D N212G.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations Y104V G128S G131P Q206L A216V.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations S9E Y104V G128S G131*.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations Y104V G128S G131P Q206L.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations Y104V G128S G131P S159E Q206L.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations S9E Y104V G128S G131P.

In another particular embodiment, the subtilase variant comprises orconsists of the set of mutations Y104V G128S G131P S159E Q206L A216V.

In a third aspect of the invention, the subtilase variant comprises orconsists of at least three mutations, for example at least fourmutations, selected from the group consisting of S9E/D, N76D/E,A88V/I/L/M, S159E/D, S204D/E, Q206L/I/V/M, L209W, N212G/A/S,A216V/I/L/M, Y217L/I/V/M F261W/N/T and Y262E/D, wherein positions arenumbered according to SEQ ID NO: 1, and wherein the variant has proteaseactivity and a sequence identity to SEQ ID NO: 1 of at least 80%, forexample at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least98%, but less than 100%.

In a particular embodiment of this aspect of the invention, thesubtilase variant comprises or consists of the substitutionsQ206L/I/V/M, N212G/A/S and A216V/I/L/M, preferably Q206L, N212G andA216V. The variant of this embodiment may further include one or moreadditional substitutions, for example one or more substitutions selectedfrom S9E/D, N76D/E, S159E/D and S204D/E, preferably S204D/E, morepreferably S204D.

In another embodiment of this aspect of the invention, the subtilasevariant comprises or consists of the substitutions N76D/E, S159E/D,Q206L/I/V/M, N212G/A/S and A216V/I/L/M, preferably N76D, S159E, Q206L,N212G and A216V.

In another embodiment, the subtilase variant comprises or consists ofthe substitutions S204D/E, Q206L/I/V/M, N212G/A/S and A216V/I/L/M,preferably S204D, Q206L, N212G and A216V.

In another embodiment, the subtilase variant comprises or consists ofthe substitutions S159E/D, Q206L/I/V/M, N212G/A/S and A216V/I/L/M,preferably S159E, Q206L, N212G and A216V.

In another embodiment, the subtilase variant comprises or consists ofthe substitutions S9E/D, S159E/D, Q206L/I/V/M and A216V/I/L/M,preferably S9E, S159E, Q206L and A216V.

In another embodiment, the subtilase variant comprises or consists ofthe substitutions N76D/E, N212G/A/S, F261W/N/T and Y262E/D, preferablyN76D, N212G, F261W and Y262E.

In another embodiment, the subtilase variant comprises or consists ofthe substitutions S204D/E, Q206L/I/V/M and N212G/A/S, preferably S204D,Q206L and N212G.

In another embodiment, the subtilase variant comprises or consists ofthe substitutions S9E/D, S204D/E and N212G/A/S, preferably S9E, S204Dand N212G.

In another embodiment, the subtilase variant comprises or consists ofthe substitutions N76D/E, A88V/I/L/M, S204D/E and N212G/A/S, preferablyN76D, A88V, S204D and N212G.

In another embodiment, the subtilase variant comprises or consists ofthe substitutions Q206L/I/V/M, F261W/N/T and Y262E/D, preferably Q206L,F261W and Y262E.

In another embodiment, the subtilase variant comprises or consists ofthe substitutions Q206L/I/V/M, L209W/N/T, A216V/I/L/M and Y217L/I/V/M,preferably Q206L, L209W, A216V and Y217L.

A further aspect of the invention relates to subtilase variantscomprising two mutations in positions selected from 9, 63, 76, 88, 104,107, 128, 131, 159, 204, 206, 209, 212, 215, 216, 261 and 262, whereinpositions are numbered according to SEQ ID NO: 1, and wherein thevariant has protease activity and a sequence identity to SEQ ID NO: 1 ofat least 80%, for example at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, or at least 98%, but less than 100%. In this aspect of theinvention, the mutations are in particular selected from the following:

-   -   the mutation in position 9 is S9E or S9D, preferably S9E;    -   the mutation in position 63 is S63G or S63A, preferably S63G;    -   the mutation in position 76 is N76D or N76E, preferably N76D;    -   the mutation in position 88 is A88V, A88I, A88L or A88M,        preferably A88V;    -   the mutation in position 104 is Y104V, Y104I, Y104L or Y104M,        preferably Y104V;    -   the mutation in position 107 is I107L, I107V, I107M, preferably        I107L;    -   the mutation in position 128 is G128S, G128A or G128T,        preferably G128S;    -   the mutation in position 131 is G131* or G131P;    -   the mutation in position 159 is S159E or S159D, preferably        S159E;    -   the mutation in position 204 is S204D or S204E, preferably        S204D;    -   the mutation in position 206 is Q206L, Q206I, Q206V or Q206M,        preferably Q206L;    -   the mutation in position 209 is L209W;    -   the mutation in position 212 is N212G, N212A or N212S,        preferably N212G;    -   the mutation in position 215 is G215A;    -   the mutation in position 216 is A216V, A216I, A216L or A216M,        preferably A216V;    -   the mutation in position 217 is Y217L, Y217I, Y217V or Y217M,        preferably Y217L;    -   the mutation in position 261 is F261W, F261N or F261Y,        preferably F261W; and/or    -   the mutation in position 262 is Y262E or Y262D, preferably        Y262E.

In one embodiment of this aspect of the invention, the variant comprisesa first mutation S9E or S9D, preferably S9E, and a second mutation S63Gor S63A, preferably S63G.

In another embodiment, the variant comprises a first mutation S9E orS9D, preferably S9E, and a second mutation N76D or N76E, preferablyN76D.

In another embodiment, the variant comprises a first mutation S9E orS9D, preferably S9E, and a second mutation A88V, A88I, A88L or A88M,preferably A88V.

In another embodiment, the variant comprises a first mutation S9E orS9D, preferably S9E, and a second mutation Y104V, Y104I, Y104L or Y104M,preferably Y104V.

In another embodiment, the variant comprises a first mutation S9E orS9D, preferably S9E, and a second mutation I107L, I107V, I107M,preferably I107L.

In another embodiment, the variant comprises a first mutation S9E orS9D, preferably S9E, and a second mutation G128S, G128A or G128T,preferably G128S.

In another embodiment, the variant comprises a first mutation S9E orS9D, preferably S9E, and a second mutation G131*.

In another embodiment, the variant comprises a first mutation S9E orS9D, preferably S9E, and a second mutation G131P.

In another embodiment, the variant comprises a first mutation S9E orS9D, preferably S9E, and a second mutation S159E or S159D, preferablyS159E.

In another embodiment, the variant comprises a first mutation S9E orS9D, preferably S9E, and a second mutation S204D or S204E, preferablyS204D.

In another embodiment, the variant comprises a first mutation S9E orS9D, preferably S9E, and a second mutation Q206L, Q206I, Q206V or Q206M,preferably Q206L.

In another embodiment, the variant comprises a first mutation S9E orS9D, preferably S9E, and a second mutation L209W.

In another embodiment, the variant comprises a first mutation S9E orS9D, preferably S9E, and a second mutation N212G, N212A or N212S,preferably N212G.

In another embodiment, the variant comprises a first mutation S9E orS9D, preferably S9E, and a second mutation G215A.

In another embodiment, the variant comprises a first mutation S9E orS9D, preferably S9E, and a second mutation A216V, A216I, A216L or A216M,preferably A216V.

In another embodiment, the variant comprises a first mutation S9E orS9D, preferably S9E, and a second mutation Y217L, Y217I, Y217V or Y217M,preferably Y217L.

In another embodiment, the variant comprises a first mutation S9E orS9D, preferably S9E, and a second mutation F261W, F261N or F261Y,preferably F261W.

In another embodiment, the variant comprises a first mutation S9E orS9D, preferably S9E, and a second mutation Y262E or Y262D, preferablyY262E.

In another embodiment, the variant comprises a first mutation S63G orS63A, preferably S63G, and a second mutation N76D or N76E, preferablyN76D.

In another embodiment, the variant comprises a first mutation S63G orS63A, preferably S63G, and a second mutation A88V, A88I, A88L or A88M,preferably A88V.

In another embodiment, the variant comprises a first mutation S63G orS63A, preferably S63G, and a second mutation Y104V, Y104I, Y104L orY104M, preferably Y104V.

In another embodiment, the variant comprises a first mutation S63G orS63A, preferably S63G, and a second mutation I107L, I107V, I107M,preferably I107L.

In another embodiment, the variant comprises a first mutation S63G orS63A, preferably S63G, and a second mutation G128S, G128A or G128T,preferably G128S.

In another embodiment, the variant comprises a first mutation S63G orS63A, preferably S63G, and a second mutation G131*.

In another embodiment, the variant comprises a first mutation S63G orS63A, preferably S63G, and a second mutation G131P.

In another embodiment, the variant comprises a first mutation S63G orS63A, preferably S63G, and a second mutation S159E or S159D, preferablyS159E.

In another embodiment, the variant comprises a first mutation S63G orS63A, preferably S63G, and a second mutation S204D or S204E, preferablyS204D.

In another embodiment, the variant comprises a first mutation S63G orS63A, preferably S63G, and a second mutation Q206L, Q206I, Q206V orQ206M, preferably Q206L.

In another embodiment, the variant comprises a first mutation S63G orS63A, preferably S63G, and a second mutation L209W.

In another embodiment, the variant comprises a first mutation S63G orS63A, preferably S63G, and a second mutation N212G, N212A or N212S,preferably N212G.

In another embodiment, the variant comprises a first mutation S63G orS63A, preferably S63G, and a second mutation G215A.

In another embodiment, the variant comprises a first mutation S63G orS63A, preferably S63G, and a second mutation A216V, A216I, A216L orA216M, preferably A216V.

In another embodiment, the variant comprises a first mutation S63G orS63A, preferably S63G, and a second mutation Y217L, Y2171, Y217V orY217M, preferably Y217L.

In another embodiment, the variant comprises a first mutation S63G orS63A, preferably S63G, and a second mutation F261W, F261N or F261Y,preferably F261W.

In another embodiment, the variant comprises a first mutation S63G orS63A, preferably S63G, and a second mutation Y262E or Y262D, preferablyY262E.

In another embodiment, the variant comprises a first mutation N76D orN76E, preferably N76D, and a second mutation A88V, A88I, A88L or A88M,preferably A88V.

In another embodiment, the variant comprises a first mutation N76D orN76E, preferably N76D, and a second mutation Y104V, Y104I, Y104L orY104M, preferably Y104V.

In another embodiment, the variant comprises a first mutation N76D orN76E, preferably N76D, and a second mutation I107L, I107V, I107M,preferably I107L.

In another embodiment, the variant comprises a first mutation N76D orN76E, preferably N76D, and a second mutation G128S, G128A or G128T,preferably G128S.

In another embodiment, the variant comprises a first mutation N76D orN76E, preferably N76D, and a second mutation G131*.

In another embodiment, the variant comprises a first mutation N76D orN76E, preferably N76D, and a second mutation G131P.

In another embodiment, the variant comprises a first mutation N76D orN76E, preferably N76D, and a second mutation S159E or S159D, preferablyS159E.

In another embodiment, the variant comprises a first mutation N76D orN76E, preferably N76D, and a second mutation S204D or S204E, preferablyS204D.

In another embodiment, the variant comprises a first mutation N76D orN76E, preferably N76D, and a second mutation Q206L, Q206I, Q206V orQ206M, preferably Q206L.

In another embodiment, the variant comprises a first mutation N76D orN76E, preferably N76D, and a second mutation L209W.

In another embodiment, the variant comprises a first mutation N76D orN76E, preferably N76D, and a second mutation N212G, N212A or N212S,preferably N212G.

In another embodiment, the variant comprises a first mutation N76D orN76E, preferably N76D, and a second mutation G215A.

In another embodiment, the variant comprises a first mutation N76D orN76E, preferably N76D, and a second mutation A216V, A216I, A216L orA216M, preferably A216V.

In another embodiment, the variant comprises a first mutation N76D orN76E, preferably N76D, and a second mutation Y217L, Y217I, Y217V orY217M, preferably Y217L.

In another embodiment, the variant comprises a first mutation N76D orN76E, preferably N76D, and a second mutation F261W, F261N or F261Y,preferably F261W.

In another embodiment, the variant comprises a first mutation N76D orN76E, preferably N76D, and a second mutation Y262E or Y262D, preferablyY262E.

In another embodiment, the variant comprises a first mutation A88V,A88I, A88L or A88M, preferably A88V, and a second mutation Y104V, Y104I,Y104L or Y104M, preferably Y104V.

In another embodiment, the variant comprises a first mutation A88V,A88I, A88L or A88M, preferably A88V, and a second mutation I107L, I107V,I107M, preferably I107L.

In another embodiment, the variant comprises a first mutation A88V,A88I, A88L or A88M, preferably A88V, and a second mutation G128S, G128Aor G128T, preferably G128S.

In another embodiment, the variant comprises a first mutation A88V,A88I, A88L or A88M, preferably A88V, and a second mutation G131*.

In another embodiment, the variant comprises a first mutation A88V,A88I, A88L or A88M, preferably A88V, and a second mutation G131P.

In another embodiment, the variant comprises a first mutation A88V,A88I, A88L or A88M, preferably A88V, and a second mutation S159E orS159D, preferably S159E.

In another embodiment, the variant comprises a first mutation A88V,A88I, A88L or A88M, preferably A88V, and a second mutation S204D orS204E, preferably S204D.

In another embodiment, the variant comprises a first mutation A88V,A88I, A88L or A88M, preferably A88V, and a second mutation Q206L, Q206I,Q206V or Q206M, preferably Q206L.

In another embodiment, the variant comprises a first mutation A88V,A88I, A88L or A88M, preferably A88V, and a second mutation L209W.

In another embodiment, the variant comprises a first mutation A88V,A88I, A88L or A88M, preferably A88V, and a second mutation N212G, N212Aor N212S, preferably N212G.

In another embodiment, the variant comprises a first mutation A88V,A88I, A88L or A88M, preferably A88V, and a second mutation G215A.

In another embodiment, the variant comprises a first mutation A88V,A88I, A88L or A88M, preferably A88V, and a second mutation A216V, A216I,A216L or A216M, preferably A216V.

In another embodiment, the variant comprises a first mutation A88V,A88I, A88L or A88M, preferably A88V, and a second mutation Y217L, Y217I,Y217V or Y217M, preferably Y217L.

In another embodiment, the variant comprises a first mutation A88V,A88I, A88L or A88M, preferably A88V, and a second mutation F261W, F261Nor F261Y, preferably F261W.

In another embodiment, the variant comprises a first mutation A88V,A88I, A88L or A88M, preferably A88V, and a second mutation Y262E orY262D, preferably Y262E.

In another embodiment, the variant comprises a first mutation Y104V,Y104I, Y104L or Y104M, preferably Y104V, and a second mutation I107L,I107V, I107M, preferably I107L.

In another embodiment, the variant comprises a first mutation Y104V,Y104I, Y104L or Y104M, preferably Y104V, and a second mutation G128S,G128A or G128T, preferably G128S.

In another embodiment, the variant comprises a first mutation Y104V,Y104I, Y104L or Y104M, preferably Y104V, and a second mutation G131*.

In another embodiment, the variant comprises a first mutation Y104V,Y104I, Y104L or Y104M, preferably Y104V, and a second mutation G131P.

In another embodiment, the variant comprises a first mutation Y104V,Y104I, Y104L or Y104M, preferably Y104V, and a second mutation S159E orS159D, preferably S159E.

In another embodiment, the variant comprises a first mutation Y104V,Y104I, Y104L or Y104M, preferably Y104V, and a second mutation S204D orS204E, preferably S204D.

In another embodiment, the variant comprises a first mutation Y104V,Y104I, Y104L or Y104M, preferably Y104V, and a second mutation Q206L,Q206I, Q206V or Q206M, preferably Q206L.

In another embodiment, the variant comprises a first mutation Y104V,Y104I, Y104L or Y104M, preferably Y104V, and a second mutation L209W.

In another embodiment, the variant comprises a first mutation Y104V,Y104I, Y104L or Y104M, preferably Y104V, and a second mutation N212G,N212A or N212S, preferably N212G.

In another embodiment, the variant comprises a first mutation Y104V,Y104I, Y104L or Y104M, preferably Y104V, and a second mutation G215A.

In another embodiment, the variant comprises a first mutation Y104V,Y104I, Y104L or Y104M, preferably Y104V, and a second mutation A216V,A216I, A216L or A216M, preferably A216V.

In another embodiment, the variant comprises a first mutation Y104V,Y104I, Y104L or Y104M, preferably Y104V, and a second mutation Y217L,Y217I, Y217V or Y217M, preferably Y217L.

In another embodiment, the variant comprises a first mutation Y104V,Y104I, Y104L or Y104M, preferably Y104V, and a second mutation F261W,F261N or F261Y, preferably F261W.

In another embodiment, the variant comprises a first mutation Y104V,Y104I, Y104L or Y104M, preferably Y104V, and a second mutation Y262E orY262D, preferably Y262E.

In another embodiment, the variant comprises a first mutation I107L,I107V, I107M, preferably I107L, and a second mutation G128S, G128A orG128T, preferably G128S.

In another embodiment, the variant comprises a first mutation I107L,I107V, I107M, preferably I107L, and a second mutation G131*.

In another embodiment, the variant comprises a first mutation I107L,I107V, I107M, preferably I107L, and a second mutation G131P.

In another embodiment, the variant comprises a first mutation I107L,I107V, I107M, preferably I107L, and a second mutation S159E or S159D,preferably S159E.

In another embodiment, the variant comprises a first mutation I107L,I107V, I107M, preferably I107L, and a second mutation S204D or S204E,preferably S204D.

In another embodiment, the variant comprises a first mutation I107L,I107V, I107M, preferably I107L, and a second mutation Q206L, Q206I,Q206V or Q206M, preferably Q206L.

In another embodiment, the variant comprises a first mutation I107L,I107V, I107M, preferably I107L, and a second mutation L209W.

In another embodiment, the variant comprises a first mutation I107L,I107V, I107M, preferably I107L, and a second mutation N212G, N212A orN212S, preferably N212G.

In another embodiment, the variant comprises a first mutation I107L,I107V, I107M, preferably I107L, and a second mutation G215A.

In another embodiment, the variant comprises a first mutation I107L,I107V, I107M, preferably I107L, and a second mutation A216V, A216I,A216L or A216M, preferably A216V.

In another embodiment, the variant comprises a first mutation I107L,I107V, I107M, preferably I107L, and a second mutation Y217L, Y217I,Y217V or Y217M, preferably Y217L.

In another embodiment, the variant comprises a first mutation I107L,I107V, I107M, preferably I107L, and a second mutation F261W, F261N orF261Y, preferably F261W.

In another embodiment, the variant comprises a first mutation I107L,I107V, I107M, preferably I107L, and a second mutation Y262E or Y262D,preferably Y262E.

In another embodiment, the variant comprises a first mutation G128S,G128A or G128T, preferably G128S, and a second mutation G131*.

In another embodiment, the variant comprises a first mutation G128S,G128A or G128T, preferably G128S, and a second mutation G131P.

In another embodiment, the variant comprises a first mutation G128S,G128A or G128T, preferably G128S, and a second mutation S159E or S159D,preferably S159E.

In another embodiment, the variant comprises a first mutation G128S,G128A or G128T, preferably G128S, and a second mutation S204D or S204E,preferably S204D.

In another embodiment, the variant comprises a first mutation G128S,G128A or G128T, preferably G128S, and a second mutation Q206L, Q206I,Q206V or Q206M, preferably Q206L.

In another embodiment, the variant comprises a first mutation G128S,G128A or G128T, preferably G128S, and a second mutation L209W.

In another embodiment, the variant comprises a first mutation G128S,G128A or G128T, preferably G128S, and a second mutation N212G, N212A orN212S, preferably N212G.

In another embodiment, the variant comprises a first mutation G128S,G128A or G128T, preferably G128S, and a second mutation G215A.

In another embodiment, the variant comprises a first mutation G128S,G128A or G128T, preferably G128S, and a second mutation A216V, A216I,A216L or A216M, preferably A216V.

In another embodiment, the variant comprises a first mutation G128S,G128A or G128T, preferably G128S, and a second mutation Y217L, Y217I,Y217V or Y217M, preferably Y217L.

In another embodiment, the variant comprises a first mutation G128S,G128A or G128T, preferably G128S, and a second mutation F261W, F261N orF261Y, preferably F261W.

In another embodiment, the variant comprises a first mutation G128S,G128A or G128T, preferably G128S, and a second mutation Y262E or Y262D,preferably Y262E.

In another embodiment, the variant comprises a first mutation G131*, anda second mutation S159E or S159D, preferably S159E.

In another embodiment, the variant comprises a first mutation G131*, anda second mutation S204D or S204E, preferably S204D.

In another embodiment, the variant comprises a first mutation G131*, anda second mutation Q206L, Q206I, Q206V or Q206M, preferably Q206L.

In another embodiment, the variant comprises a first mutation G131*, anda second mutation L209W.

In another embodiment, the variant comprises a first mutation G131*, anda second mutation N212G, N212A or N212S, preferably N212G.

In another embodiment, the variant comprises a first mutation G131*, anda second mutation G215A.

In another embodiment, the variant comprises a first mutation G131*, anda second mutation A216V, A216I, A216L or A216M, preferably A216V.

In another embodiment, the variant comprises a first mutation G131*, anda second mutation Y217L, Y217I, Y217V or Y217M, preferably Y217L.

In another embodiment, the variant comprises a first mutation G131*, anda second mutation F261W, F261N or F261Y, preferably F261W.

In another embodiment, the variant comprises a first mutation G131*, anda second mutation Y262E or Y262D, preferably Y262E.

In another embodiment, the variant comprises a first mutation G131P, anda second mutation S159E or S159D, preferably S159E.

In another embodiment, the variant comprises a first mutation G131P, anda second mutation S204D or S204E, preferably S204D.

In another embodiment, the variant comprises a first mutation G131P, anda second mutation Q206L, Q206I, Q206V or Q206M, preferably Q206L.

In another embodiment, the variant comprises a first mutation G131P, anda second mutation L209W.

In another embodiment, the variant comprises a first mutation G131P, anda second mutation N212G, N212A or N212S, preferably N212G.

In another embodiment, the variant comprises a first mutation G131P, anda second mutation G215A.

In another embodiment, the variant comprises a first mutation G131P, anda second mutation A216V, A216I, A216L or A216M, preferably A216V.

In another embodiment, the variant comprises a first mutation G131P, anda second mutation Y217L, Y217I, Y217V or Y217M, preferably Y217L.

In another embodiment, the variant comprises a first mutation G131P, anda second mutation F261W, F261N or F261Y, preferably F261W.

In another embodiment, the variant comprises a first mutation G131P, anda second mutation Y262E or Y262D, preferably Y262E.

In another embodiment, the variant comprises a first mutation S159E orS159D, preferably S159E, and a second mutation S204D or S204E,preferably S204D.

In another embodiment, the variant comprises a first mutation S159E orS159D, preferably S159E, and a second mutation Q206L, Q206I, Q206V orQ206M, preferably Q206L.

In another embodiment, the variant comprises a first mutation S159E orS159D, preferably S159E, and a second mutation L209W.

In another embodiment, the variant comprises a first mutation S159E orS159D, preferably S159E, and a second mutation N212G, N212A or N212S,preferably N212G.

In another embodiment, the variant comprises a first mutation S159E orS159D, preferably S159E, and a second mutation G215A.

In another embodiment, the variant comprises a first mutation S159E orS159D, preferably S159E, and a second mutation A216V, A216I, A216L orA216M, preferably A216V.

In another embodiment, the variant comprises a first mutation S159E orS159D, preferably S159E, and a second mutation Y217L, Y217I, Y217V orY217M, preferably Y217L.

In another embodiment, the variant comprises a first mutation S159E orS159D, preferably S159E, and a second mutation F261W, F261N or F261Y,preferably F261W.

In another embodiment, the variant comprises a first mutation S159E orS159D, preferably S159E, and a second mutation Y262E or Y262D,preferably Y262E.

In another embodiment, the variant comprises a first mutation S204D orS204E, preferably S204D, and a second mutation Q206L, Q206I, Q206V orQ206M, preferably Q206L.

In another embodiment, the variant comprises a first mutation S204D orS204E, preferably S204D, and a second mutation L209W.

In another embodiment, the variant comprises a first mutation S204D orS204E, preferably S204D, and a second mutation N212G, N212A or N212S,preferably N212G.

In another embodiment, the variant comprises a first mutation S204D orS204E, preferably S204D, and a second mutation G215A.

In another embodiment, the variant comprises a first mutation S204D orS204E, preferably S204D, and a second mutation A216V, A216I, A216L orA216M, preferably A216V.

In another embodiment, the variant comprises a first mutation S204D orS204E, preferably S204D, and a second mutation Y217L, Y217I, Y217V orY217M, preferably Y217L.

In another embodiment, the variant comprises a first mutation S204D orS204E, preferably S204D, and a second mutation F261W, F261N or F261Y,preferably F261W.

In another embodiment, the variant comprises a first mutation S204D orS204E, preferably S204D, and a second mutation Y262E or Y262D,preferably Y262E.

In another embodiment, the variant comprises a first mutation Q206L,Q206I, Q206V or Q206M, preferably Q206L, and a second mutation L209W.

In another embodiment, the variant comprises a first mutation Q206L,Q206I, Q206V or Q206M, preferably Q206L, and a second mutation N212G,N212A or N212S, preferably N212G.

In another embodiment, the variant comprises a first mutation Q206L,Q206I, Q206V or Q206M, preferably Q206L, and a second mutation G215A.

In another embodiment, the variant comprises a first mutation Q206L,Q206I, Q206V or Q206M, preferably Q206L, and a second mutation A216V,A216I, A216L or A216M, preferably A216V.

In another embodiment, the variant comprises a first mutation Q206L,Q206I, Q206V or Q206M, preferably Q206L, and a second mutation Y217L,Y217I, Y217V or Y217M, preferably Y217L.

In another embodiment, the variant comprises a first mutation Q206L,Q206I, Q206V or Q206M, preferably Q206L, and a second mutation F261W,F261N or F261Y, preferably F261W.

In another embodiment, the variant comprises a first mutation Q206L,Q206I, Q206V or Q206M, preferably Q206L, and a second mutation Y262E orY262D, preferably Y262E.

In another embodiment, the variant comprises a first mutation L209W, anda second mutation N212G, N212A or N212S, preferably N212G.

In another embodiment, the variant comprises a first mutation L209W, anda second mutation G215A.

In another embodiment, the variant comprises a first mutation L209W, anda second mutation A216V, A216I, A216L or A216M, preferably A216V.

In another embodiment, the variant comprises a first mutation L209W, anda second mutation Y217L, Y217I, Y217V or Y217M, preferably Y217L.

In another embodiment, the variant comprises a first mutation L209W, anda second mutation F261W, F261N or F261Y, preferably F261W.

In another embodiment, the variant comprises a first mutation L209W, anda second mutation Y262E or Y262D, preferably Y262E.

In another embodiment, the variant comprises a first mutation N212G,N212A or N212S, preferably N212G, and a second mutation G215A.

In another embodiment, the variant comprises a first mutation N212G,N212A or N212S, preferably N212G, and a second mutation A216V, A216I,A216L or A216M, preferably A216V.

In another embodiment, the variant comprises a first mutation N212G,N212A or N212S, preferably N212G, and a second mutation Y217L, Y217I,Y217V or Y217M, preferably Y217L.

In another embodiment, the variant comprises a first mutation N212G,N212A or N212S, preferably N212G, and a second mutation F261W, F261N orF261Y, preferably F261W.

In another embodiment, the variant comprises a first mutation N212G,N212A or N212S, preferably N212G, and a second mutation Y262E or Y262D,preferably Y262E.

In another embodiment, the variant comprises a first mutation G215A, anda second mutation A216V, A216I, A216L or A216M, preferably A216V.

In another embodiment, the variant comprises a first mutation G215A, anda second mutation Y217L, Y217I, Y217V or Y217M, preferably Y217L.

In another embodiment, the variant comprises a first mutation G215A, anda second mutation F261W, F261N or F261Y, preferably F261W.

In another embodiment, the variant comprises a first mutation G215A, anda second mutation Y262E or Y262D, preferably Y262E.

In another embodiment, the variant comprises a first mutation A216V,A216I, A216L or A216M, preferably A216V, and a second mutation Y217L,Y217I, Y217V or Y217M, preferably Y217L.

In another embodiment, the variant comprises a first mutation A216V,A216I, A216L or A216M, preferably A216V, and a second mutation F261W,F261N or F261Y, preferably F261W.

In another embodiment, the variant comprises a first mutation A216V,A216I, A216L or A216M, preferably A216V, and a second mutation Y262E orY262D, preferably Y262E.

In another embodiment, the variant comprises a first mutation Y217L,Y217I, Y217V or Y217M, preferably Y217L, and a second mutation F261W,F261N or F261Y, preferably F261W.

In another embodiment, the variant comprises a first mutation Y217L,Y217I, Y217V or Y217M, preferably Y217L, and a second mutation Y262E orY262D, preferably Y262E.

In another embodiment, the variant comprises a first mutation F261W,F261N or F261Y, preferably F261W, and a second mutation Y262E or Y262D,preferably Y262E.

Any of the variants of the invention may have a sequence identity to theamino acid sequence of SEQ ID NO: 1 of at least 85%, for example atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, or at least 98%, but less than100%.

The number of alterations in the variants of the present inventioncompared to SEQ ID NO: 1 may, for example, be in the range of 1-20,e.g., 1-10 or 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations.

In addition to the amino acid alterations specifically disclosed herein,a protease variant of the invention may comprise additional alterationsat one or more other positions. These additional alterations may be of aminor nature, that is conservative amino acid substitutions orinsertions that do not significantly affect the folding and/or activityof the protein; small deletions, typically of 1-30 amino acids; smallamino- or carboxyl-terminal extensions, such as an amino-terminalmethionine residue; a small linker peptide of up to 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, in The Proteins, Academic Press, New York. Commonconservative substitution groups include, but are not limited to: G=A=S;I=V=L=M; D=E; Y=F; and N=Q (where e.g. “G=A=S” means that these threeamino acids may be substituted for each other).

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for protease activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide. ForBPN′ (SEQ ID NO: 1) the catalytic triad comprising the amino acids S221,H64, and D32 is considered essential for protease activity of theenzyme.

The polypeptides of the invention may consist of 220 to 330 amino acids,such as 240 to 300, 260 to 290 or 270 to 280 amino acids.

Method for Stabilizing a Subtilase Variant

Another aspect of the invention relates to a method for stabilizing asubtilase variant, the method comprising introducing into a parentsubtilase having protease activity and at least 80% sequence identity toSEQ ID NO: 1, for example at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% sequence identity, atleast three mutations selected from:

-   -   (i) L209W in combination with two or more substitutions selected        from S63G/A, G215A and Y217L/I/V/M;    -   (ii) Y104V/I/L/M and G128S/A/T in combination with at least one        mutation, e.g. two, three or more mutations, selected from        S9E/D, S63G/A, N76D/E, I107L/V/M, G131*, G131P, S159E/D,        S204D/E, Q206L/IN/M, N212G/A/S, G215A, A216V/l/L/M and        Y217L/IN/M;    -   (iii) S9E/D in combination with at least two substitutions        selected from N76D/E, S204D/E and N212G/A/S;    -   (iv) N76D/E in combination with at least two substitutions        selected from S9E/D, A88V/I/L/M, S159E/D, S204D/E, Q206L/I/V/M,        N212G/A/S, A216V/I/L/M, F261W/N/Y and Y262E/D;    -   (v) Q206L/I/V/M in combination with at least two substitutions        selected from S9E/D, N76D/E, S159E/D, S204D/E, L209W/N/Y,        N212G/A/S, A216V/I/L/M, Y217L/I/V/M, F261W/N/Y and Y262E/D; and    -   (vi) S204D/E, N212G/A/S and A216V/I/L/M, preferably in        combination with at least one substitution selected from S9E/D,        S159E/D and Q206L/I/V/M.

In the case of (i) in the method for stabilizing a subtilase variantabove, the mutations may e.g. comprise S63G, L209W, G215A and Y217L; andoptionally one or more additional mutations such one, two, three or moremutations selected from S9E/D, N76D/E, Y104V/I/L/M, G128S/A/T, G131*,G131P, S204D/E, Q206L/I/V/M, A216V/I/L/M, F261W/N/Y and Y262E/D; e.g.one, two, three or more mutations selected from S9E, N76D, Y104V, G128S,G131* or G131P, S204D, Q206L, A216V, F261W and Y262E.

In the case of (ii) in the method for stabilizing a subtilase variantabove, the mutations may e.g. comprise Y104V and G128S in combinationwith at least one mutation, e.g. two, three or more mutations, selectedfrom S9E, S63G, N76D, I107L, G131* or G131P, S159E, S204D, Q206L, N212G,G215A, A216V and Y217L.

In the case of (iii) in the method for stabilizing a subtilase variantabove, the mutations may e.g. comprise S9E in combination with at leasttwo substitutions selected from N76D, S204D and N212G.

In the case of (iv) in the method for stabilizing a subtilase variantabove, the mutations may e.g. comprise N76D in combination with at leasttwo substitutions selected from S9E, A88V, S159E, S204D, Q206L, N212G,A216V, F261W and Y262E.

In the case of (v) in the method for stabilizing a subtilase variantabove, the mutations may e.g. comprise Q206L in combination with atleast two substitutions selected from S9E, N76D, S159E, S204D, L209W,N212G, A216V, Y217L, F261W and Y262E.

In the case of (vi) in the method for stabilizing a subtilase variantabove, the mutations may e.g. comprise S204D, Q206L, N212G and A216V.

It will be understood that the method for stabilizing a subtilasevariant is meant to encompass introducing into the parent subtilase anycombination of mutations disclosed above under the heading “Variants”.

The variants of the invention, or the variants stabilized by the methodabove, preferably have an increased storage stability in a detergentcomposition compared to the subtilase of SEQ ID NO: 1.

Storage Stability

In one aspect, any of the protease variants of the invention, orprotease variants stabilized by the method above, have an increasedstorage stability in a detergent composition compared to the subtilaseof SEQ ID NO: 1. Storage stability may suitably be measured as describedin storage stability assay I in Example 2 herein, and/or as measured asdescribed in storage stability assay II in Example 3 herein.

Preferably, the protease variants of the invention, or protease variantsstabilized by the method above, have an increased storage stability in adetergent composition compared to the subtilase of SEQ ID NO: 1 of atleast 25%, preferably at least 50%, more preferably at least 75%, suchas at least 100%, when measured for 24 hours as described in storagestability assay I in Example 2 herein, and/or as measured as describedin storage stability assay II in Example 3 herein.

Parent Subtilases

The parent subtilase of a variant of the invention will typically be aprotease that has at least 75% identity with the sequence of subtilisinBPN′ from Bacillus amyloliquefaciens, which has the sequence of SEQ IDNO: 1. In preferred embodiments, the parent subtilase may have at least80% identity to SEQ ID NO: 1, such as at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% identity with SEQID NO: 1. Alternatively, the parent subtilase may have a sequence thatcomprises of consists of SEQ ID NO: 1.

The parent may thus, for example, have the sequence of a wild-typesubtilase such as BPN′ (SEQ ID NO: 1), or alternatively may be a variantof BPN′. The parent may also be a related subtilase, e.g. from the S8Afamily having at least 75% sequence identity to SEQ ID NO: 1 asindicated above.

In one embodiment, the amino acid sequence of the parent may for examplediffer by up to 20 amino acids, such as up to 10 amino acids, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, from the polypeptide of SEQID NO: 1.

The parent subtilase may also be a fragment of the polypeptide of SEQ IDNO: 1 that has protease activity, or an allelic variant of thepolypeptide of SEQ ID NO: 1.

The parent subtilase may be obtained from a microorganism of anysuitable genus, in particular from a suitable bacteria genus. The parentsubtilase is thus typically a bacterial subtilase. For example, theparent may be a Gram-positive bacterial polypeptide such as a Bacillus,Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus,Oceanobacillus, Staphylococcus, Streptococcus or Streptomyces subtilase,or a Gram-negative bacterial polypeptide such as a Campylobacter, E.coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter,Neisseria, Pseudomonas, Salmonella, or Ureaplasma subtilase.

In one embodiment, the parent is obtained from a species of Bacillus.The parent may thus e.g. be a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis or Bacillus thuringiensissubtilase.

In one embodiment, the parent is a Bacillus amyloliquefaciens protease,e.g. the protease of SEQ ID NO: 1.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, compost, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to a method for obtaining a subtilasevariant, the method comprising (a) providing a host cell comprising apolynucleotide encoding a variant of a parent protease having three ormore mutations, e.g. four or more mutations, compared to SEQ ID NO: 1,wherein the variant has protease activity and a sequence identity to SEQID NO: 1 of at least 80%, for example at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity, and wherein the mutations are selected from:

-   -   (i) L209W in combination with two or more substitutions selected        from S63G/A, G215A and Y217L/I/V/M;    -   (ii) Y104V/I/L/M and G128S/A/T in combination with at least one        mutation selected from S9E/D, S63G/A, N76D/E, I107L/V/M, G131*,        G131P, S159E/D, S204D/E, Q206L/I/V/M, N212G/A/S, G215A,        A216V/I/L/M and Y217L/I/V/M;    -   (iii) S9E/D in combination with at least two substitutions        selected from N76D/E, S204D/E and N212G/A/S;    -   (iv) N76D/E in combination with at least two substitutions        selected from S9E/D, A88V/I/L/M, S159E/D, S204D/E, Q206L/I/V/M,        N212G/A/S, A216V/I/L/M, F261W/N/Y and Y262E/D;    -   (v) Q206L/I/V/M in combination with at least two substitutions        selected from S9E/D, N76D/E, S159E/D, S204D/E, L209W/N/Y,        N212G/A/S, A216V/I/L/M, Y217L/I/V/M, F261W/N/Y and Y262E/D; and    -   (vi) S204D/E, N212G/A/S and A216V/I/L/M, preferably in        combination with at least one substitution selected from S9E/D,        S159E/D and Q206L/I/V/M;        (b) cultivating the host cell under conditions suitable for        expression of the variant; and        (c) recovering the variant.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, DNA shuffling,etc. For information on use of these mutagenesis techniques, see e.g. WO2017/207762.

In the case of (i) in the method for obtaining a subtilase variantabove, the mutations may e.g. comprise S63G, L209W, G215A and Y217L; andoptionally one or more additional mutations such one, two, three or moremutations selected from S9E/D, N76D/E, Y104V/I/L/M, G128S/A/T, G131*,G131P, S204D/E, Q206L/I/V/M, A216V/I/L/M, F261W/N/Y and Y262E/D; e.g.one, two, three or more mutations selected from S9E, N76D, Y104V, G128S,G131* or G131P, S204D, Q206L, A216V, F261W and Y262E.

In the case of (ii) in the method for obtaining a subtilase variantabove, the mutations may e.g. comprise Y104V and G128S in combinationwith at least one mutation, e.g. two, three or more mutations, selectedfrom S9E, S63G, N76D, I107L, G131* or G131P, S159E, S204D, Q206L, N212G,G215A, A216V and Y217L.

In the case of (iii) in the method for obtaining a subtilase variantabove, the mutations may e.g. comprise S9E in combination with at leasttwo substitutions selected from N76D, S204D and N212G.

In the case of (iv) in the method for obtaining a subtilase variantabove, the mutations may e.g. comprise N76D in combination with at leasttwo substitutions selected from S9E, A88V, S159E, S204D, Q206L, N212G,A216V, F261W and Y262E.

In the case of (v) in the method for obtaining a subtilase variantabove, the mutations may e.g. comprise Q206L in combination with atleast two substitutions selected from S9E, N76D, S159E, S204D, L209W,N212G, A216V, Y217L, F261W and Y262E.

In the case of (vi) in the method for obtaining a subtilase variantabove, the mutations may e.g. comprise S204D, Q206L, N212G and A216V.

It will be understood that the method for obtaining a subtilase variantis meant to encompass expression and recovery of variants having anycombination of mutations disclosed above under the heading “Variants”.

Polynucleotides

The present invention also relates to polynucleotides encoding a variantof the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor and optimize expression of a variant. Techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart. These include, e.g., the use of control sequences such aspromoters, transcription terminators, mRNA stabilizer regions downstreamof a promoter and upstream of the coding sequence, signal peptide codingregions, propeptide coding sequences and regulatory sequences. Forfurther information, see e.g. WO 2017/207762.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or morerestriction sites to allow for insertion or substitution of thepolynucleotide encoding the variant at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

For information on expression vectors, see e.g. WO 2017/207762.

Host Cells

The present invention also relates to recombinant host cells comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier.

The term “host cell” encompasses any progeny of a parent cell that isnot identical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the variant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell will typically be a Gram-positive orGram-negative bacterium, such as a Gram-positive bacterium selected fromBacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus andStreptomyces, or a Gram-negative bacterium selected from Campylobacter,E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter,Neisseria, Pseudomonas, Salmonella and Ureaplasma.

The bacterial host cell may e.g. be a Bacillus cell selected fromBacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,Bacillus subtilis and Bacillus thuringiensis cells.

For information on suitable host cells, see e.g. WO 2017/207762.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for expression of the variant; and (b) recoveringthe variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid-state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants with protease activity, and may be recoveredand purified using methods known in the art. See e.g. WO 2017/207762 forfurther information.

Compositions

The invention also relates to a composition comprising a subtilasevariant of the invention, e.g. a detergent or cleaning composition.

The invention also relates to a composition comprising a subtilasevariant of the invention and further comprising: one or more detergentcomponents; and/or one or more additional enzymes. In a preferredembodiment, the composition is a detergent composition comprising one ormore detergent components.

The present invention also relates to a composition comprising asubtilase variant of the present invention and further comprising one ormore additional enzymes selected from the group consisting of amylases,catalases, cellulases (e.g., endoglucanases), cutinases,haloperoxygenases, lipases, mannanases, pectinases, pectin lyases,peroxidases, proteases, xanthanases, lichenases and xyloglucanases, orany mixture thereof.

A detergent composition may e.g. be in the form of a bar, a homogeneoustablet, a tablet having two or more layers, a pouch having one or morecompartments, a regular or compact powder, a granule, a paste, a gel, ora regular, compact or concentrated liquid.

The invention also relates to use of a composition of the present in acleaning process, such as laundry or hard surface cleaning such as dishwash.

The choice of additional components for a detergent composition iswithin the skill of the artisan and includes conventional ingredients,including the exemplary non-limiting components set forth below. Thechoice of components may include, for fabric care, the consideration ofthe type of fabric to be cleaned, the type and/or degree of soiling, thetemperature at which cleaning is to take place, and the formulation ofthe detergent product.

In a particular embodiment, a detergent composition comprises asubtilase variant of the invention and one or more detergent components,such as surfactants, hydrotropes, builders, co-builders, chelators orchelating agents, bleaching system or bleach components, polymers,fabric hueing agents, fabric conditioners, foam boosters, sudssuppressors, dispersants, dye transfer inhibitors, fluorescent whiteningagents, perfume, optical brighteners, bactericides, fungicides, soilsuspending agents, soil release polymers, anti-redeposition agents,enzyme inhibitors or stabilizers, enzyme activators, antioxidants, andsolubilizers.

In one embodiment, one or more of the detergent components may benon-naturally occurring detergent components. In another embodiment, allof the detergent components may be non-naturally occurring detergentcomponents.

In one embodiment, the subtilase variant of the invention may be addedto a detergent composition in an amount corresponding to 0.01-200 mg ofenzyme protein per liter of wash liquor, preferably 0.05-50 mg of enzymeprotein per liter of wash liquor, in particular 0.1-10 mg of enzymeprotein per liter of wash liquor.

An automatic dish wash (ADW) composition may for example include0.001%-30%, such as 0.01%-20%, such as 0.1-15%, such as 0.5-10% ofenzyme protein by weight of the composition.

A granulated composition for laundry may for example include 0.001%-20%,such as 0.01%-10%, such as 0.05%-5% of enzyme protein by weight of thecomposition.

A liquid composition for laundry may for example include 0.0001%-10%,such as 0.001-7%, such as 0.1%-5% of enzyme protein by weight of thecomposition.

The enzymes such as the subtilase variant of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, for example, WO92/19709 and WO 92/19708 or the variants according to the invention maybe stabilized using peptide aldehydes or ketones such as described in WO2005/105826 and WO 2009/118375.

The subtilase variants of the invention may be formulated in liquidlaundry compositions such as a liquid laundry compositions compositioncomprising:

-   -   a) at least 0.01 mg of active subtilase variant per litre        detergent,    -   b) 2 wt % to 60 wt % of at least one surfactant    -   c) 5 wt % to 50 wt % of at least one builder

The detergent composition may be formulated into a granular detergentfor laundry. Such detergent may comprise;

-   -   a) at least 0.01 mg of active protease variant per gram of        composition    -   b) anionic surfactant, preferably 5 wt % to 50 wt %    -   c) nonionic surfactant, preferably 1 wt % to 8 wt %    -   d) builder, preferably 5 wt % to 40 wt %, such as carbonates,        zeolites, phosphate builder, calcium sequestering builders or        complexing agents.

Although components mentioned below are categorized by general headeraccording to a particular functionality, this is not to be construed asa limitation, as a component may comprise additional functionalities aswill be appreciated by the person skilled in the art.

Surfactants

The detergent composition may comprise one or more surfactants, whichmay be anionic and/or cationic and/or non-ionic and/or semi-polar and/orzwitterionic, or a mixture thereof. In a particular embodiment, thedetergent composition includes a mixture of one or more nonionicsurfactants and one or more anionic surfactants. The surfactant(s) istypically present at a level of from about 0.1% to 60% by weight, suchas about 1% to about 40%, or about 3% to about 20%, or about 3% to about10%. The surfactant(s) is chosen based on the desired cleaningapplication, and includes any conventional surfactant(s) known in theart. Any surfactant known in the art for use in detergents may beutilized. Surfactants lower the surface tension in the detergent, whichallows the stain being cleaned to be lifted and dispersed and thenwashed away.

When included therein, the detergent will usually contain from about 1%to about 40% by weight, such as from about 5% to about 30%, includingfrom about 5% to about 15%, or from about 20% to about 25% of an anionicsurfactant. Non-limiting examples of anionic surfactants includesulfates and sulfonates, in particular, linear alkylbenzenesulfonates(LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS),phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates,alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonatesand disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate(SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS),alcohol ethersulfates (AES or AEOS or FES, also known as alcoholethoxysulfates or fatty alcohol ether sulfates), secondaryalkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates,sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methylesters (alpha-SFMe or SES) including methyl ester sulfonate (MES),alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid(DTSA), fatty acid derivatives of amino acids, diesters and monoestersof sulfo-succinic acid or soap, and combinations thereof.

When included therein, the detergent will usually contain from about 0%to about 10% by weight of a cationic surfactant. Non-limiting examplesof cationic surfactants include alklydimethylethanolamine quat (ADMEAQ),cetyltrimethylammonium bromide (CTAB), dimethyldistearylammoniumchloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternaryammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, andcombinations thereof.

When included therein, the detergent will usually contain from about0.2% to about 40% by weight of a non-ionic surfactant, for example fromabout 0.5% to about 30%, in particular from about 1% to about 20%, fromabout 3% to about 10%, such as from about 3% to about 5%, or from about8% to about 12%. Non-limiting examples of non-ionic surfactants includealcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylatedfatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such asethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenolethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides(APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fattyacid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides(EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine(glucamides, GA, or fatty acid glucamide, FAGA), as well as productsavailable under the trade names SPAN and TWEEN, and combinationsthereof.

When included therein, the detergent will usually contain from about 0%to about 10% by weight of a semipolar surfactant. Non-limiting examplesof semipolar surfactants include amine oxides (AO) such asalkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxide andN-(tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, fatty acidalkanolamides and ethoxylated fatty acid alkanolamides, and combinationsthereof.

When included therein, the detergent will usually contain from about 0%to about 10% by weight of a zwitterionic surfactant. Non-limitingexamples of zwitterionic surfactants include betaine,alkyldimethylbetaine, sulfobetaine, and combinations thereof.

Builders and Co-Builders

The detergent composition may contain about 0-65% by weight, such asabout 5% to about 45% of a detergent builder or co-builder, or a mixturethereof. In a dish wash deteregent, the level of builder is typically40-65%, particularly 50-65%. Builders and chelators soften, e.g., thewash water by removing the metal ions form the liquid. The builderand/or co-builder may particularly be a chelating agent that formswater-soluble complexes with Ca and Mg. Any builder and/or co-builderknown in the art for use in laundry detergents may be utilized.Non-limiting examples of builders include zeolites, diphosphates(pyrophosphates), triphosphates such as sodium triphosphate (STP orSTPP), carbonates such as sodium carbonate, soluble silicates such assodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst),ethanolamines such as 2-aminoethan-1-ol (MEA), diethanolamine (DEA, alsoknown as iminodiethanol), triethanolamine (TEA, also known as2,2′,2″-nitrilotriethanol), and carboxymethyl inulin (CMI), andcombinations thereof.

The detergent composition may also contain 0-20% by weight, such asabout 5% to about 10%, of a detergent co-builder, or a mixture thereof.The detergent composition may include a co-builder alone, or incombination with a builder, for example a zeolite builder. Non-limitingexamples of co-builders include homopolymers of polyacrylates orcopolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylicacid/maleic acid) (PAA/PMA). Further non-limiting examples includecitrate, chelators such as aminocarboxylates, aminopolycarboxylates andphosphonates, and alkyl- or alkenylsuccinic acid. Additional specificexamples include 2,2′,2″-nitrilotriacetic acid (NTA),ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N′-disuccinicacid (EDDS), methylglycinediacetic acid (MGDA), glutamicacid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid(HEDP), ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA),diethylenetriaminepentakis (methylenephosphonic acid) (DTPMPA or DTMPA),N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoaceticacid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), asparticacid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid(SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL),N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid(MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid(SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diaceticacid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilicacid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) andsulfomethyl-N,N-diacetic acid (SMDA),N-(2-hydroxyethyl)-ethylidenediamine-N,N,N′-triacetate (HEDTA),diethanolglycine (DEG), diethylenetriamine penta(methylenephosphonicacid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), andcombinations and salts thereof. Further exemplary builders and/orco-builders are described in, e.g., WO 2009/102854 and U.S. Pat. No.5,977,053.

The subtilase variants of the invention may also be formulated into adish wash composition, preferably an automatic dish wash composition(ADW), comprising:

-   -   a) at least 0.01 mg of active protease variant according to the        invention, and    -   b) 10-50 wt % builder preferably selected from citric acid,        methylglycine-N,N-diacetic acid (MGDA) and/or glutamic        acid-N,N-diacetic acid (GLDA) and mixtures thereof, and    -   c) at least one bleach component.

Bleaching Systems

The detergent may contain 0-50% by weight, such as about 0.1% to about25%, of a bleaching system. Bleach systems remove discolor often byoxidation, and many bleaches also have strong bactericidal properties,and are used for disinfecting and sterilizing. Any bleaching systemknown in the art for use in laundry detergents may be utilized. Suitablebleaching system components include bleaching catalysts, photobleaches,bleach activators, sources of hydrogen peroxide such as sodiumpercarbonate and sodium perborates, preformed peracids and mixturesthereof. Suitable preformed peracids include, but are not limited to,peroxycarboxylic acids and salts, percarbonic acids and salts, perimidicacids and salts, peroxymonosulfuric acids and salts, for example, Oxone(R), and mixtures thereof. Non-limiting examples of bleaching systemsinclude peroxide-based bleaching systems, which may comprise, forexample, an inorganic salt, including alkali metal salts such as sodiumsalts of perborate (usually mono- or tetra-hydrate), percarbonate,persulfate, perphosphate, persilicate salts, in combination with aperacid-forming bleach activator. The term bleach activator is meantherein as a compound which reacts with peroxygen bleach like hydrogenperoxide to form a peracid. The peracid thus formed constitutes theactivated bleach. Suitable bleach activators to be used herein includethose belonging to the class of esters amides, imides or anhydrides.Suitable examples are tetracetylethylene diamine (TAED), sodium4-[(3,5,5-trimethylhexanoyl)oxy]benzene sulfonate (ISONOBS), diperoxydodecanoic acid, 4-(dodecanoyloxy)benzenesulfonate (LOBS),4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS),4-(nonanoyloxy)-benzenesulfonate (NOBS), and/or those disclosed in WO98/17767. A particular family of bleach activators of interest wasdisclosed in EP 624154 and particularly preferred in that family isacetyl triethyl citrate (ATC). ATC or a short chain triglyceride liketriacetin has the advantage that it is environmentally friendly as iteventually degrades into citric acid and alcohol. Furthermore, acetyltriethyl citrate and triacetin have good hydrolytic stability in theproduct upon storage and are efficient bleach activators. Finally, ATCprovides a good building capacity to the laundry additive.Alternatively, the bleaching system may comprise peroxyacids of, forexample, the amide, imide, or sulfone type. The bleaching system mayalso comprise peracids such as 6-(phthalimido)peroxyhexanoic acid (PAP).The bleaching system may also include a bleach catalyst or a booster.

Some non-limiting examples of bleach catalysts that may be used in thecompositions of the present invention include manganese oxalate,manganese acetate, manganese-collagen, cobalt-amine catalysts andmanganese triazacyclononane (MnTACN) catalysts; particularly preferredare complexes of manganese with 1,4,7-trimethyl-1,4,7-triazacyclononane(Me3-TACN) or 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me4-TACN), inparticular Me3-TACN, such as the dinuclear manganese complex[(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2, and[2,2′,2″-nitrilotris(ethane-1,2-diylazanylylidene-κN-methanylylidene)triphenolato-κ3O]manganese(III).The bleach catalysts may also be other metal compounds, such as iron orcobalt complexes.

In some embodiments, the bleach component may be an organic catalystselected from the group consisting of organic catalysts having thefollowing formula:

-   -   (iii) and mixtures thereof; wherein each R¹ is independently a        branched alkyl group containing from 9 to 24 carbons or linear        alkyl group containing from 11 to 24 carbons, preferably each R¹        is independently a branched alkyl group containing from 9 to 18        carbons or linear alkyl group containing from 11 to 18 carbons,        more preferably each R¹ is independently selected from the group        consisting of 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl,        2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl,        iso-nonyl, iso-decyl, iso-tridecyl and iso-pentadecyl. Other        exemplary bleaching systems are described, e.g., in WO        2007/087258, WO 2007/087244, WO 2007/087259 and WO 2007/087242.        Suitable photobleaches may for example be sulfonated zinc        phthalocyanine.

Hydrotropes

A hydrotrope is a compound that solubilizes hydrophobic compounds inaqueous solutions (or oppositely, polar substances in a non-polarenvironment). Typically, hydrotropes have both hydrophilic andhydrophobic characters (so-called amphiphilic properties as known fromsurfactants); however, the molecular structures of hydrotropes generallydo not favour spontaneous self-aggregation, see, e.g., review by Hodgdonand Kaler, 2007, Current Opinion in Colloid & Interface Science 12:121-128. Hydrotropes do not display a critical concentration above whichself-aggregation occurs as found for surfactants and lipids formingmiceller, lamellar or other well defined meso-phases. Instead, manyhydrotropes show a continuous-type aggregation process where the sizesof aggregates grow as concentration increases. However, many hydrotropesalter the phase behaviour, stability, and colloidal properties ofsystems containing substances of polar and non-polar character,including mixtures of water, oil, surfactants, and polymers. Hydrotropesare classically used across industries from pharma, personal care andfood to technical applications. Use of hydrotropes in detergentcompositions allows for example more concentrated formulations ofsurfactants (as in the process of compacting liquid detergents byremoving water) without inducing undesired phenomena such as phaseseparation or high viscosity.

The detergent may contain 0-5% by weight, such as about 0.5 to about 5%,or about 3% to about 5%, of a hydrotrope. Any hydrotrope known in theart for use in detergents may be utilized. Non-limiting examples ofhydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonate(STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS),sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers,sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodiumethylhexyl sulfate, and combinations thereof.

Polymers

The detergent may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2%or 0.2-1% of a polymer. Any polymer known in the art for use indetergents may be utilized. The polymer may function as a co-builder asmentioned above, or may provide antiredeposition, fiber protection, soilrelease, dye transfer inhibition, grease cleaning and/or anti-foamingproperties. Some polymers may have more than one of the above-mentionedproperties and/or more than one of the below-mentioned motifs. Exemplarypolymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol)(PVA), poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) orpoly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine),carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA/PMA,poly-aspartic acid, and lauryl methacrylate/acrylic acid copolymers,hydrophobically modified CMC (HM-CMC) and silicones, copolymers ofterephthalic acid and oligomeric glycols, copolymers of poly(ethyleneterephthalate) and poly(oxyethene terephthalate) (PET-POET), PVP,poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO)and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplarypolymers include sulfonated polycarboxylates, polyethylene oxide andpolypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Otherexemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of theabove-mentioned polymers are also contemplated.

Fabric Hueing Agents

The detergent compositions of the present invention may also includefabric hueing agents such as dyes or pigments, which when formulated indetergent compositions can deposit onto a fabric when the fabric iscontacted with a wash liquor comprising the detergent compositions andthus altering the tint of the fabric through absorption/reflection ofvisible light. Fluorescent whitening agents emit at least some visiblelight. In contrast, fabric hueing agents alter the tint of a surface asthey absorb at least a portion of the visible light spectrum. Suitablefabric hueing agents include dyes and dye-clay conjugates, and may alsoinclude pigments. Suitable dyes include small molecule dyes andpolymeric dyes. Suitable small molecule dyes include small molecule dyesselected from the group consisting of dyes falling into the Colour Index(C.I.) classifications of Direct Blue, Direct Red, Direct Violet, AcidBlue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, ormixtures thereof, for example as described in WO 2005/003274, WO2005/003275, WO 2005/003276 and EP 1876226 (hereby incorporated byreference). The detergent composition preferably comprises from about0.00003 wt. % to about 0.2 wt. %, from about 0.00008 wt. % to about 0.05wt. %, or even from about 0.0001 wt. % to about 0.04 wt. % fabric hueingagent. The composition may comprise from 0.0001 wt % to 0.2 wt. % fabrichueing agent, this may be especially preferred when the composition isin the form of a unit dose pouch. Suitable hueing agents are alsodisclosed in, e.g., WO 2007/087257 and WO 2007/087243.

Additional Enzymes

The detergent additive as well as the detergent composition may compriseone or more (additional) enzymes such as an amylase, arabinase,carbohydrase, cellulase (e.g., endoglucanase), cutinase, galactanase,haloperoxygenase, lipase, mannanase, oxidase, e.g., laccase and/orperoxidase, pectinase, pectin lyase, protease, xylanase, xanthanase orxyloglucanase.

The properties of the selected enzyme(s) should be compatible with theselected detergent (e.g. pH-optimum, compatibility with other enzymaticand non-enzymatic ingredients, etc.).

Cellulases

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178,5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 495257, EP 531372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 531315, U.S. Pat. Nos. 5,457,046, 5,686,593,5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Examples of cellulases exhibiting endo-beta-1,4-glucanase activity (EC3.2.1.4) are described in WO 02/99091.

Other examples of cellulases include the family 45 cellulases describedin WO 96/29397, and especially variants thereof having substitution,insertion and/or deletion at one or more of the positions correspondingto the following positions in SEQ ID NO: 8 of WO 02/99091: 2, 4, 7, 8,10, 13, 15, 19, 20, 21, 25, 26, 29, 32, 33, 34, 35, 37, 40, 42, 42a, 43,44, 48, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 70, 72, 76, 79, 80, 82,84, 86, 88, 90, 91, 93, 95, 95d, 95h, 95j, 97, 100, 101, 102, 103, 113,114, 117, 119, 121, 133, 136, 137, 138, 139, 140a, 141, 143a, 145, 146,147, 150e, 150j, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160c,160e, 160k, 161, 162, 164, 165, 168, 170, 171, 172, 173, 175, 176, 178,181, 183, 184, 185, 186, 188, 191, 192, 195, 196, 200, and/or 20,preferably selected among P19A, G20K, Q44K, N48E, Q119H or Q146 R.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

Proteases

The composition may comprise one or more additional proteases includingthose of bacterial, fungal, plant, viral or animal origin, e.g.,vegetable or microbial origin. Microbial origin is preferred. Chemicallymodified or protein engineered mutants are included. It may be analkaline protease, such as a serine protease or a metalloprotease. Aserine protease may for example be of the S1 family, such as trypsin, orthe S8 family such as subtilisin. A metalloprotease may for example be athermolysin from, e.g., family M4 or other metalloprotease such as thosefrom M5, M7 or M8 families.

Examples of metalloproteases are the neutral metalloproteases asdescribed in WO 2007/044993 (Genencor Int.) such as those derived fromBacillus amyloliquefaciens.

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase®Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®,Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra,Neutrase®, Everlase® and Esperase® (Novozymes A/S), those sold under thetradename Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®,Purafect MAO, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, FN2®,FN3®, FN4®, Excellase®, Eraser®, Opticlean® and Optimase®(Danisco/DuPont), Axapem™ (Gist-Brocades N.V.), BLAP (sequence shown inFIG. 29 of U.S. Pat. No. 5,352,604) and variants hereof (Henkel AG) andKAP (Bacillus alkalophilus subtilisin) from Kao.

Lipases and Cutinases

Suitable lipases and cutinases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutant enzymes areincluded. Examples include lipase from Thermomyces, e.g., from T.lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP 305216, cutinase from Humicola, e.g., H. insolens (WO96/13580), lipase from strains of Pseudomonas (some of these now renamedto Burkholderia), e.g., P. alcaligenes or P. pseudoalcaligenes (EP218272), P. cepacia (EP 331376), P. sp. strain SD705 (WO 95/06720 & WO96/27002), P. wisconsinensis (WO 96/12012), GDSL-type Streptomyceslipases (WO 2010/065455), cutinase from Magnaporthe grisea (WO2010/107560), cutinase from Pseudomonas mendocina (U.S. Pat. No.5,389,536), lipase from Thermobifida fusca (WO 2011/084412), Geobacillusstearothermophilus lipase (WO 2011/084417), lipase from Bacillussubtilis (WO 2011/084599), and lipase from Streptomyces griseus (WO2011/150157) and S. pristinaespiralis (WO 2012/137147).

Other examples are lipase variants such as those described in EP 407225,WO 92/05249, WO 94/01541, WO 94/25578, WO 95/14783, WO 95/30744, WO95/35381, WO 95/22615, WO 96/00292, WO 97/04079, WO 97/07202, WO00/34450, WO 00/60063, WO 01/92502, WO 2007/87508 and WO 2009/109500.

Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) andLipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to asacyltransferases or perhydrolases, e.g., acyltransferases with homologyto Candida antarctica lipase A (WO 2010/111143), acyltransferase fromMycobacterium smegmatis (WO 2005/056782), perhydrolases from the CE 7family (WO 2009/067279), and variants of the M. smegmatis perhydrolasein particular the S54V variant used in the commercial product GentlePower Bleach from Huntsman Textile Effects Pte Ltd (WO 2010/100028).

Amylases

Suitable amylases which can be used together with the subtilase variantsof the invention may be an alpha-amylase or a glucoamylase and may be ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, alpha-amylasesobtained from Bacillus, e.g., a special strain of Bacilluslicheniformis, described in more detail in GB 1,296,839.

Suitable amylases include amylases having SEQ ID NO: 2 in WO 95/10603 orvariants having 90% sequence identity to SEQ ID NO: 3 thereof. Preferredvariants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQID NO: 4 of WO 99/19467, such as variants with substitutions in one ormore of the following positions: 15, 23, 105, 106, 124, 128, 133, 154,156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243,264, 304, 305, 391, 408, and 444.

Different suitable amylases include amylases having SEQ ID NO: 6 in WO02/10355 or variants thereof having 90% sequence identity to SEQ ID NO:6. Preferred variants of SEQ ID NO: 6 are those having a deletion inpositions 181 and 182 and a substitution in position 193.

Other amylases which are suitable are hybrid alpha-amylase comprisingresidues 1-33 of the alpha-amylase derived from B. amyloliquefaciensshown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of the B.licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 orvariants having 90% sequence identity thereof. Preferred variants ofthis hybrid alpha-amylase are those having a substitution, a deletion oran insertion in one of more of the following positions: G48, T49, G107,H156, A181, N190, M197, 1201, A209 and Q264. Most preferred variants ofthe hybrid alpha-amylase comprising residues 1-33 of the alpha-amylasederived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having thesubstitutions:

-   -   M197T;    -   H156Y+A181T+N190F+A209V+Q264S; or    -   G48A+T491+G107A+H156Y+A181T+N190F+1201F+A209V+Q264S.

Other suitable amylases are amylases having the sequence of SEQ ID NO: 6in WO 99/19467 or variants thereof having 90% sequence identity to SEQID NO: 6. Preferred variants of SEQ ID NO: 6 are those having asubstitution, a deletion or an insertion in one or more of the followingpositions: R181, G182, H183, G184, N195, I206, E212, E216 and K269.Particularly preferred amylases are those having deletion in positionsR181 and G182, or positions H183 and G184.

Additional amylases which can be used are those having SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/23873 or variantsthereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, adeletion or an insertion in one or more of the following positions: 140,181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using SEQID 2 of WO 96/23873 for numbering. More preferred variants are thosehaving a deletion in two positions selected from 181, 182, 183 and 184,such as 181 and 182, 182 and 183, or positions 183 and 184. Mostpreferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7are those having a deletion in positions 183 and 184 and a substitutionin one or more of positions 140, 195, 206, 243, 260, 304 and 476.

Other amylases which can be used are amylases having SEQ ID NO: 2 of WO2008/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO 2008/153815 or 90% sequenceidentity to SEQ ID NO: 10 in WO 01/66712. Preferred variants of SEQ IDNO: 10 in WO 01/66712 are those having a substitution, a deletion or aninsertion in one of more of the following positions: 176, 177, 178, 179,190, 201, 207, 211 and 264.

Further suitable amylases are amylases having SEQ ID NO: 2 of WO2009/061380 or variants having 90% sequence identity to SEQ ID NO: 2thereof. Preferred variants of SEQ ID NO: 2 are those having atruncation of the C-terminus and/or a substitution, a deletion or aninsertion in one of more of the following positions: Q87, Q98, S125,N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243,N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferredvariants of SEQ ID NO: 2 are those having the substitution in one ofmore of the following positions: Q87E,R, Q98R, S125A, N128C, T131I,T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R,R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180and/or S181 or of T182 and/or G183. Most preferred amylase variants ofSEQ ID NO: 2 are those having the substitutions:

-   -   N128C+K178L+T182G+Y305R+G475K;    -   N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;    -   S125A+N128C+K178L+T182G+Y305R+G475K; or    -   S125A+N1280+T131I+T165I+K178L+T182G+Y305R+G475K,        wherein the variants are C-terminally truncated and optionally        further comprise a substitution at position 243 and/or a        deletion at position 180 and/or position 181.

Further suitable amylases are amylases having SEQ ID NO: 1 of WO2013/184577 or variants having 90% sequence identity to SEQ ID NO: 1thereof. Preferred variants of SEQ ID NO: 1 are those having asubstitution, a deletion or an insertion in one of more of the followingpositions: K176, R178, G179, T180, G181, E187, N192, M199, 1203, S241,R458, T459, D460, G476 and G477. More preferred variants of SEQ ID NO: 1are those having the substitution in one of more of the followingpositions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S,D460T, G476K and G477K and/or a deletion in position R178 and/or S179 orof T180 and/or G181. Most preferred amylase variants of SEQ ID NO: 1comprise the substitutions:

-   -   E187P+I203Y+G476K    -   E187P+I203Y+R458N+T459S+D460T+G476K        and optionally further comprise a substitution at position 241        and/or a deletion at position 178 and/or position 179.

Further suitable amylases are amylases having SEQ ID NO: 1 of WO2010/104675 or variants having 90% sequence identity to SEQ ID NO: 1thereof. Preferred variants of SEQ ID NO: 1 are those having asubstitution, a deletion or an insertion in one of more of the followingpositions: N21, D97, V128 K177, R179, S180, I181, G182, M200, L204,E242, G477 and G478.

More preferred variants of SEQ ID NO: 1 are those having thesubstitution in one of more of the following positions: N21D, D97N,V128I K177L, M200L, L204YF, E242QA, G477K and G478K and/or a deletion inposition R179 and/or S180 or of I181 and/or G182. Most preferred amylasevariants of SEQ ID NO: 1 comprise the substitutions N21D+D97N+V128I, andoptionally further comprise a substitution at position 200 and/or adeletion at position 180 and/or position 181.

Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 in WO01/66712 or a variant having at least 90% sequence identity to SEQ IDNO: 12. Preferred amylase variants are those having a substitution, adeletion or an insertion in one of more of the following positions ofSEQ ID NO: 12 in WO 01/66712: R28, R118, N174; R181, G182, D183, G184,G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320,H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484.Particularly preferred amylases include variants having a deletion ofD183 and G184 and having the substitutions R118K, N195F, R320K andR458K, and a variant additionally having substitutions in one or moreposition selected from the group: M9, G149, G182, G186, M202, T257,Y295, N299, M323, E345 and A339, most preferred a variant thatadditionally has substitutions in all these positions.

Other examples are amylase variants such as those described in WO2011/098531, WO 2013/001078 and WO 2013/001087. Commercially availableamylases are Duramyl™, Termamyl™, Fungamyl™, Stainzyme™, StainzymePlus™, Natalase™, Liquozyme X and BAN™ (from Novozymes A/S), andRapidase™, Purastar™/Effectenz™, Powerase, Preferenz S1000, PreferenzS100 and Preferenz S110 (from Genencor International Inc./DuPont).

Peroxidases/Oxidases

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g., from C. cinereus, and variants thereof as thosedescribed in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

Adjunct Materials

Any detergent components known in the art for use in laundry detergentsmay also be utilized. Other optional detergent components includeanti-corrosion agents, anti-shrink agents, anti-soil redepositionagents, anti-wrinkling agents, bactericides, binders, corrosioninhibitors, disintegrants/disintegration agents, dyes, enzymestabilizers (including boric acid, borates, CMC, and/or polyols such aspropylene glycol), fabric conditioners including clays,fillers/processing aids, fluorescent whitening agents/opticalbrighteners, foam boosters, foam (suds) regulators, perfumes,soil-suspending agents, softeners, suds suppressors, tarnish inhibitors,and wicking agents, either alone or in combination. Any ingredient knownin the art for use in laundry detergents may be utilized. The choice ofsuch ingredients is well within the skill of the artisan.

Dispersants: The detergent compositions of the present invention canalso contain dispersants. In particular powdered detergents may comprisedispersants. Suitable water-soluble organic materials include the homo-or co-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms. Suitable dispersants are for exampledescribed in Powdered Detergents, Surfactant science series volume 71,Marcel Dekker, Inc.

Dye Transfer Inhibiting Agents: The detergent compositions of thepresent invention may also include one or more dye transfer inhibitingagents. Suitable polymeric dye transfer inhibiting agents include, butare not limited to, polyvinylpyrrolidone polymers, polyamine N-oxidepolymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole,polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. Whenpresent in a subject composition, the dye transfer inhibiting agents maybe present at levels from about 0.0001% to about 10%, from about 0.01%to about 5% or even from about 0.1% to about 3% by weight of thecomposition.

Fluorescent whitening agent: The detergent compositions of the presentinvention will preferably also contain additional components that maytint articles being cleaned, such as fluorescent whitening agent oroptical brighteners. Where present the brightener is preferably at alevel of about 0.01% to about 05%. Any fluorescent whitening agentsuitable for use in a laundry detergent composition may be used in thecomposition of the present invention. The most commonly used fluorescentwhitening agents are those belonging to the classes ofdiaminostilbene-sulphonic acid derivatives, diarylpyrazoline derivativesand bisphenyl-distyryl derivatives. Examples of thediaminostilbene-sulphonic acid derivative type of fluorescent whiteningagents include the sodium salts of:4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2′-disulphonate; 4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2.2′-disulphonate;4,4′-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)stilbene-2,2′-disulphonate;4,4′-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino) stilbene-2,2′-disulphonate and2-(stilbyl-4″-naptho-1.,2′:4,5)-1,2,3-trizole-2″-sulphonate. Preferredfluorescent whitening agents are Tinopal DMS and Tinopal CBS availablefrom Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium saltof 4,4′-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino) stilbenedisulphonate. Tinopal CBS is the disodium salt of2,2′-bis-(phenyl-styryl) disulphonate. Also preferred are fluorescentwhitening agents is the commercially available Parawhite KX, supplied byParamount Minerals and Chemicals, Mumbai, India. Other fluorescerssuitable for use in the invention include the 1-3-diaryl pyrazolines andthe 7-alkylaminocoumarins. Suitable fluorescent brightener levelsinclude lower levels of from about 0.01, from 0.05, from about 0.1 oreven from about 0.2 wt. % to upper levels of 0.5 or even 0.75 wt. %.

Soil release polymers: The detergent compositions of the presentinvention may also include one or more soil release polymers which aidthe removal of soils from fabrics such as cotton and polyester basedfabrics, in particular the removal of hydrophobic soils from polyesterbased fabrics. The soil release polymers may for example be nonionic oranionic terephthalte based polymers, polyvinyl caprolactam and relatedcopolymers, vinyl graft copolymers, polyester polyamides see for exampleChapter 7 in Powdered Detergents, Surfactant science series volume 71,Marcel Dekker, Inc. Another type of soil release polymers areamphiphilic alkoxylated grease cleaning polymers comprising a corestructure and a plurality of alkoxylate groups attached to that corestructure. The core structure may comprise a polyalkylenimine structureor a polyalkanolamine structure as described in detail in WO 2009/087523(hereby incorporated by reference). Furthermore, random graftco-polymers are suitable soil release polymers Suitable graftco-polymers are described in more detail in WO 2007/138054, WO2006/108856 and WO 2006/113314 (hereby incorporated by reference). Othersoil release polymers are substituted polysaccharide structuresespecially substituted cellulosic structures such as modified cellulosederiviatives such as those described in EP 1867808 or WO 03/040279 (bothare hereby incorporated by reference). Suitable cellulosic polymersinclude cellulose, cellulose ethers, cellulose esters, cellulose amidesand mixtures thereof. Suitable cellulosic polymers include anionicallymodified cellulose, nonionically modified cellulose, cationicallymodified cellulose, zwitterionically modified cellulose, and mixturesthereof. Suitable cellulosic polymers include methyl cellulose, carboxymethyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, hydroxylpropyl methyl cellulose, ester carboxy methyl cellulose, and mixturesthereof.

Anti-redeposition agents: The detergent compositions of the presentinvention may also include one or more anti-redeposition agents such ascarboxymethylcellulose (CMC), polyvinyl alcohol (PVA),polyvinylpyrrolidone (PVP), polyoxyethylene and/or polyethyleneglycol(PEG), homopolymers of acrylic acid, copolymers of acrylic acid andmaleic acid, and ethoxylated polyethyleneimines. The cellulose basedpolymers described under soil release polymers above may also functionas anti-redeposition agents.

Other suitable adjunct materials include, but are not limited to,anti-shrink agents, anti-wrinkling agents, bactericides, binders,carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foamregulators, hydrotropes, perfumes, pigments, sod suppressors, solvents,and structurants for liquid detergents and/or structure elasticizingagents.

Formulation of Detergent Products

The detergent enzyme(s), i.e. a subtilase variant of the invention andoptionally one or more additional enzymes, may be included in adetergent composition by adding separate additives containing one ormore enzymes, or by adding a combined additive comprising all of theseenzymes. A detergent additive comprising one or more enzymes can beformulated, for example, as a granulate, liquid, slurry, etc. Preferreddetergent additive formulations include granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a homogenous tablet, a tablet having two or morelayers, a pouch having one or more compartments, a regular or compactpowder, a granule, a paste, a gel, or a regular, compact or concentratedliquid. There are a number of detergent formulation forms such as layers(same or different phases), pouches, as well as forms for machine dosingunit.

Pouches can be configured as single or multiple compartments. It can beof any form, shape and material which is suitable for hold thecomposition, e.g., without allowing the release of the composition fromthe pouch prior to water contact. The pouch is made from water solublefilm which encloses an inner volume. The inner volume can be dividedinto compartments of the pouch. Preferred films are polymeric materials,preferably polymers which are formed into a film or sheet. Preferredpolymers, copolymers or derivates thereof are selected frompolyacrylates, and water-soluble acrylate copolymers, methyl cellulose,carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethylcellulose, hydroxypropyl methyl cellulose, maltodextrin,polymethacrylates, most preferably polyvinyl alcohol copolymers andhydroxypropyl methyl cellulose (HPMC). Preferably the level of polymerin the film for example PVA is at least about 60%. The preferred averagemolecular weight will typically be about 20,000 to about 150,000. Filmscan also be of blend compositions comprising hydrolytically degradableand water-soluble polymer blends such as polylactide and polyvinylalcohol (known under the Trade reference M8630 as sold by Chris CraftIn. Prod. of Gary, Ind., US) plus plasticizers like glycerol, ethyleneglycerol, propylene glycol, sorbitol and mixtures thereof. The pouchescan comprise a solid laundry detergent composition or part componentsand/or a liquid cleaning composition or part components separated by thewater-soluble film. The compartment for liquid components can bedifferent in composition than compartments containing solids. See, e.g.,US 2009/0011970.

Detergent ingredients can be separated physically from each other bycompartments in water dissolvable pouches or in different layers oftablets. Thereby negative storage interaction between components can beavoided. Different dissolution profiles of each of the compartments canalso give rise to delayed dissolution of selected components in the washsolution.

A liquid or gel detergent which is not unit dosed may be aqueous,typically containing at least 20% by weight and up to 95% water, such asup to about 70% water, up to about 65% water, up to about 55% water, upto about 45% water, up to about 35% water. Other types of liquids,including without limitation, alkanols, amines, diols, ethers andpolyols may be included in an aqueous liquid or gel. An aqueous liquidor gel detergent may contain from 0-30% organic solvent. A liquid or geldetergent may be non-aqueous.

Laundry Soap Bars

The enzymes of the invention may be added to laundry soap bars and usedfor hand washing laundry, fabrics and/or textiles. The term laundry soapbar includes laundry bars, soap bars, combo bars, syndet bars anddetergent bars. The types of bar usually differ in the type ofsurfactant they contain, and the term laundry soap bar includes thosecontaining soaps from fatty acids and/or synthetic soaps. The laundrysoap bar has a physical form which is solid and not a liquid, gel or apowder at room temperature. The term solid is defined as a physical formwhich does not significantly change over time, i.e., if a solid object(e.g., laundry soap bar) is placed inside a container, the solid objectdoes not change to fill the container it is placed in. The bar is asolid typically in bar form but can be in other solid shapes such asround or oval.

The laundry soap bar may contain one or more additional enzymes,protease inhibitors such as peptide aldehydes (or hydrosulfite adduct orhemiacetal adduct), boric acid, borate, borax and/or phenylboronic acidderivatives such as 4-formylphenylboronic acid, one or more soaps orsynthetic surfactants, polyols such as glycerine, pH controllingcompounds such as fatty acids, citric acid, acetic acid and/or formicacid, and/or a salt of a monovalent cation and an organic anion whereinthe monovalent cation may be for example Na⁺, K⁺ or NH₄ ⁺ and theorganic anion may be for example formate, acetate, citrate or lactatesuch that the salt of a monovalent cation and an organic anion may be,for example, sodium formate.

The laundry soap bar may also contain complexing agents like EDTA andHEDP, perfumes and/or different type of fillers, surfactants, e.g.,anionic synthetic surfactants, builders, polymeric soil release agents,detergent chelators, stabilizing agents, fillers, dyes, colorants, dyetransfer inhibitors, alkoxylated polycarbonates, suds suppressers,structurants, binders, leaching agents, bleaching activators, clay soilremoval agents, anti-redeposition agents, polymeric dispersing agents,brighteners, fabric softeners, perfumes and/or other compounds known inthe art.

The laundry soap bar may be processed in conventional laundry soap barmaking equipment such as but not limited to: mixers, plodders, e.g., atwo-stage vacuum plodder, extruders, cutters, logo-stampers, coolingtunnels and wrappers. The invention is not limited to preparing thelaundry soap bars by any single method. The premix of the invention maybe added to the soap at different stages of the process. For example,the premix containing a soap, an enzyme, optionally one or moreadditional enzymes, a protease inhibitor, and a salt of a monovalentcation and an organic anion may be prepared and the mixture is thenplodded. The enzyme and optional additional enzymes may be added at thesame time as the protease inhibitor for example in liquid form. Besidesthe mixing step and the plodding step, the process may further comprisethe steps of milling, extruding, cutting, stamping, cooling and/orwrapping.

Granular Detergent Formulations

Enzymes in the form of granules, comprising an enzyme-containing coreand optionally one or more coatings, are commonly used in granular(powder) detergents. Various methods for preparing the core arewell-known in the art and include, for example, a) spray drying of aliquid enzyme-containing solution, b) production of layered productswith an enzyme coated as a layer around a pre-formed inert coreparticle, e.g. using a fluid bed apparatus, c) absorbing an enzyme ontoand/or into the surface of a pre-formed core, d) extrusion of anenzyme-containing paste, e) suspending an enzyme-containing powder inmolten wax and atomization to result in prilled products, f) mixergranulation by adding an enzyme-containing liquid to a dry powdercomposition of granulation components, g) size reduction ofenzyme-containing cores by milling or crushing of larger particles,pellets, etc., and h) fluid bed granulation. The enzyme-containing coresmay be dried, e.g. using a fluid bed drier or other known methods fordrying granules in the feed or enzyme industry, to result in a watercontent of typically 0.1 -10% w/w water.

The enzyme-containing cores are optionally provided with a coating toimprove storage stability and/or to reduce dust formation. One type ofcoating that is often used for enzyme granulates for detergents is asalt coating, typically an inorganic salt coating, which may e.g. beapplied as a solution of the salt using a fluid bed. Other coatingmaterials that may be used are, for example, polyethylene glycol (PEG),methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA). Thegranules may contain more than one coating, for example a salt coatingfollowed by an additional coating of a material such as PEG, MHPC orPVA.

For further information on enzyme granules and production thereof, seeWO 2013/007594 as well as e.g. WO 2009/092699, EP 1705241, EP 1382668,WO 2007/001262, U.S. Pat. No. 6,472,364, WO 2004/074419 and WO2009/102854.

Uses

The present invention is also directed to methods for using thesubtilase variants according to the invention or compositions thereof inlaundering of textile and fabrics, such as household laundry washing andindustrial laundry washing.

The invention is also directed to methods for using the variantsaccording to the invention or compositions thereof in cleaning hardsurfaces such as floors, tables, walls, roofs etc. as well as surfacesof hard objects such as cars (car wash) and dishes (dish wash).

The subtilase variants of the present invention may be added to and thusbecome a component of a detergent composition. Thus, one aspect of theinvention relates to the use of a subtilase variant in a cleaningprocess such as laundering and/or hard surface cleaning.

A detergent composition of the present invention may be formulated, forexample, as a hand or machine laundry detergent composition including alaundry additive composition suitable for pre-treatment of stainedfabrics and a rinse added fabric softener composition, or be formulatedas a detergent composition for use in general household hard surfacecleaning operations, or be formulated for hand or machine dishwashingoperations.

In a specific aspect, the present invention provides a detergentadditive comprising a polypeptide of the present invention as describedherein.

The cleaning process or the textile care process may for example be alaundry process, a dishwashing process or cleaning of hard surfaces suchas bathroom tiles, floors, table tops, drains, sinks and washbasins.Laundry processes can for example be household laundering, but may alsobe industrial laundering. Furthermore, the invention relates to aprocess for laundering of fabrics and/or garments, where the processcomprises treating fabrics with a washing solution containing adetergent composition and at least one protease variant of theinvention. The cleaning process or a textile care process can forexample be carried out in a machine washing process or in a manualwashing process. The washing solution can for example be an aqueouswashing solution containing a detergent composition.

The last few years there has been an increasing interest in replacingcomponents in detergents that are derived from petrochemicals withrenewable biological components such as enzymes and polypeptides withoutcompromising the wash performance. When the components of detergentcompositions change, new enzyme activities or new enzymes havingalternative and/or improved properties compared to the previously useddetergent enzymes such as proteases, lipases and amylases may be neededto achieve a similar or improved wash performance when compared to thetraditional detergent compositions.

The invention further concerns the use of subtilase variants of theinvention in a proteinaceous stain removing process. The proteinaceousstains may be stains such as food stains, e.g., baby food, cocoa, egg ormilk, or other stains such as sebum blood, ink or grass, or acombination hereof.

Washing Method

The present invention relates to a method of cleaning a fabric, adishware or hard surface with a detergent composition comprising aprotease variant of the invention.

A preferred embodiment concerns a method of cleaning, the methodcomprising the steps of: contacting an object with a detergentcomposition comprising a protease variant of the invention underconditions suitable for cleaning the object. In a preferred embodimentthe detergent composition is used in a laundry or a dish wash process.

Still another embodiment relates to a method for removing stains fromfabric or dishware which comprises contacting the fabric or dishwarewith a composition comprising a protease of the invention underconditions suitable for cleaning the object. In the method of cleaningof the invention, the object being cleaned may be any suitable objectsuch as a textile or a hard surface such as dishware or a floor, table,wall, etc.

Also contemplated are compositions and methods of treating fabrics(e.g., to desize a textile) using one or more of the protease of theinvention. The protease can be used in any fabric-treating method whichis well known in the art (see, e.g., U.S. Pat. No. 6,077,316). Forexample, in one aspect, the feel and appearance of a fabric is improvedby a method comprising contacting the fabric with a protease in asolution. In one aspect, the fabric is treated with the solution underpressure.

The detergent compositions of the present invention are suited for usein laundry and hard surface applications, including dish wash.Accordingly, the present invention includes a method for laundering afabric or washing dishware. The method comprises the steps of contactingthe fabric/dishware to be cleaned with a solution comprising thedetergent composition according to the invention. The fabric maycomprise any fabric capable of being laundered in normal consumer useconditions. The dishware may comprise any dishware such as crockery,cutlery, ceramics, plastics such as melamine, metals, china, glass andacrylics. The solution preferably has a pH from about 5.5 to about 11.5.The compositions may be employed at concentrations from about 100 ppm,preferably 500 ppm to about 15,000 ppm in solution. The watertemperatures typically range from about 5° C. to about 95° C., includingabout 10° C., about 15° C., about 20° C., about 25° C., about 30° C.,about 35° C., about 40° C., about 45° C., about 50° C., about 55° C.,about 60° C., about 65° C., about 70° C., about 75° C., about 80° C.,about 85° C. and about 90° C. The water to fabric ratio is typicallyfrom about 1:1 to about 30:1.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents and proteaseinhibitors, e.g., a polyol such as propylene glycol or glycerol, a sugaror sugar alcohol, different salts such as NaCl; KCl; lactic acid, formicacid, boric acid, or a boric acid derivative, e.g., an aromatic borateester, or a phenyl boronic acid derivative such as 4-formylphenylboronic acid, or a peptide aldehyde such as di-, tri- or tetrapeptidealdehydes or aldehyde analogues (either of the form B1-B0-R wherein, Ris H, CH3, CX3, CHX2, or CH2X (X=halogen), B0 is a single amino acidresidue (preferably with an optionally substituted aliphatic or aromaticside chain); and B1 consists of one or more amino acid residues(preferably one, two or three), optionally comprising an N-terminalprotection group, or as described in WO 2009/118375, WO 98/13459) or aprotease inhibitor of the protein type such as RASI, BASI, WASI(bifunctional alpha-amylase/subtilisin inhibitors of rice, barley andwheat) or Cl2 or SSI. The composition may be formulated as described in,e.g., WO 92/19709, WO 92/19708 and U.S. Pat. No. 6,472,364. In someembodiments, the enzymes employed herein are stabilized by the presenceof water-soluble sources of zinc (II), calcium (II) and/or magnesium(II) ions in the finished compositions that provide such ions to theenzymes, as well as other metal ions (e.g., barium (II), scandium (II),iron (II), manganese (II), aluminum (III), Tin (II), cobalt (II), copper(II), Nickel (II), and oxovanadium (IV)).

In some preferred embodiments, the detergent compositions providedherein are typically formulated such that, during use in aqueouscleaning operations, the wash water has a pH of from about 5.0 to about12.5, or in alternative embodiments from about 5.0 to about 11.5, or inalternative embodiments, even from about 6.0 to about 10.5. In somepreferred embodiments, granular or liquid laundry products areformulated to have a pH from about 6 to about 8. Techniques forcontrolling pH at recommended usage levels include the use of buffers,alkalis, acids, etc., and are well known to those skilled in the art.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Example 1: Preparation of Polypeptides

Mutation and introduction of expression cassettes into Bacillus subtiliswas performed by standard methods known in the art. All DNAmanipulations were performed by PCR (e.g. as described by Sambrook etal.; Molecular Cloning; Cold Spring Harbor Laboratory Press) usingstandard methods known to the skilled person.

Recombinant B. subtilis constructs encoding subtilase polypeptides wereinoculated into and cultivated in a complex medium (TBgly) underantibiotic selection for 24 h at 37° C. Shake flasks containing a richmedia (PS-1: 100 g/L Sucrose (Danisco cat. no. 109-0429), 40 g/L crustsoy (soy bean flour), 10 g/L Na₂HPO₄.12H₂O (Merck cat. no. 106579), 0.1ml/L Dowfax63N10 (Dow) were inoculated in a ratio of 1:100 with theovernight culture. Shake flask cultivation was performed for 4 days at30° C. shaking at 270 rpm.

Recombinant B. subtilis colonies encoding subtilase polypeptides weretransferred into and cultivated in a complex medium (TBgly) underantibiotic selection for 24 h at 37° C. 24-Well deep well platescontaining 3 ml TBgly were inoculated 1:100 with overnight culture.Cultivation was performed for 4 days at 30° C. with agitation at 220rpm. For harvest, cells were sedimented by centrifugation and thesupernatant was sterilized using 0.2 μm 96-well filter plates (PallCorporation).

Example 2: Storage Stability Assay I—Stability of Variants in CultureSupernatants

Proteolytic activity can be determined by a method employingSuc-AAPF-PNA as the substrate. Suc-AAPF-PNA, or Suc-Ala-Ala-Pro-Phe-pNA,is an abbreviation forN-Succinyl-Alanine-Alanine-Proline-Phenylalanine-p-Nitroanilide, and isa blocked peptide which can be cleaved by endoproteases. Followingcleavage, a free PNA molecule is liberated which has a yellow color andthus can be measured by visible spectrophotometry at wavelength 405 nm.

Sterile filtered culture supernatants of variants of the inventionprepared as described above were tested undiluted and diluted 1:1 with0.01% Triton X-100. For each variant, 2 wells with each concentrationwere included. 30 μl protease sample was mixed with 270 μl Model Aliquid detergent (see Table 1 below) in the well of a microtiter plate(detergent plate, Nunc U96 PP 0.5 ml) using a magnetic bar (on a Zephyrpipetting station (Caliper LifeSciences) for 30 min). 20 μl of thismixture was then transferred to another microtiter plate and mixed with150 μl 0.1 M Tris pH 8.6. 30 μl of this dilution was transferred to anew microtiter plate, and after addition of 70 μl substrate solution(0.72 mg/ml Suc-Ala-Ala-Pro-Phe-pNA (Bachem L-1400) in 0.1 M Tris pH8.6) the activity of the unstressed sample was determined from theinitial slope of the increase in measured absorbance at 405 nm (measuredevery 20 sec for 5 min on a SpectraMax Plus instrument). After sealing,the detergent plate was incubated at 40° C. in an Eppendorf Thermomixer(no shaking). After 3-4 and 21-23 hours incubation, samples of 20 μlwere withdrawn and residual activity of the samples stressed bydetergent and heat was measured as with the initial unstressed activity.

The decrease in protease activity during incubation with detergent wasassumed to be exponential. Half-lives (T½) were calculated from linearregression of Log(Activity) versus incubation time, and half-lifeimprovement factors (T½ IF) were calculated as the half-life of proteasevariant relative to the half-life of the wild-type BPN′ protease (SEQ IDNO: 1) as the reference.

Table 2 below provides the half-life improvement factor for variants ofthe invention having the indicated mutations compared to the wild-typeBPN′ protease with SEQ ID NO: 1. It may be seen that all of the testedvariants provide an improved half-life in the detergent compared to thewild-type protease.

TABLE 1 Composition of Model A detergent Content of active Ingredientcomponent (% w/w) (C10-C13) alkylbenzene sulfonic acid 11.6 Sodiumlauryl ether sulfate 4.9 Soy fatty acid 2.8 Coco fatty acid 2.8 Alcoholethoxylate 11.0 Sodium hydroxide 1.8 Ethanol 2.7 Propane-2-ol 0.3Propane-1,2-diol 6.0 Glycerol 1.7 Triethanolamine 3.3 Sodium formate 1.0Sodium citrate 2.0 DTMPA-Na7 0.2 Copoly(acrylic acid/maleic acid),sodium salt 0.18 Phenoxyethanol 0.5 Water

TABLE 2 Storage Stability. T½ IF: Half- life improvement factor relativeto SEQ ID NO: 1 Mutations T½ IF S63G Q206L L209W G215A A216V Y217L 5.0S63G S204D L209W G215A Y217L 4.9 S63G N76D L209W G215A Y217L 4.5 N76DY104V G128S G131* N212G 4.0 S9E S63G L209W G215A Y217L 3.7 N76D S159EQ206L N212G A216V 3.2 Q206L N212G A216V 3.1 S63G Q206L L209W G215A Y217L3.1 S204D Q206L N212G A216V 3.0 N76D Y104V G128S G131P 2.7 S159E Q206LN212G A216V 2.6 S63G L209W G215A Y217L 2.5 S9E S159E Q206L A216V 2.3S63G Y104V G128S G131* L209W G215A Y217L 2.2 N76D Y104V G128S G131* 2.2S63G Y104V G128S G131P L209W G215A Y217L 2.1 Y104V G128S G131* Q206LA216V 2.1 Y104V G128S G131* S159E Q206L A216V 2.1 N76D Y104V G128S G131PN212G 2.0 Y104V I107L G128S G131* Q206L 2.0 N76D N212G F261W Y262E 1.8S204D Q206L N212G 1.8 S9E S204D N212G 1.8 Y104V G128S G131P Q206L A216V1.7 S63G L209W G215A Y217L F261W Y262E 1.7 Y104V G128S G131P S204D N212G1.6 S9E Y104V G128S G131* 1.6 N76D A88V S204D N212G 1.5 Y104V G128SG131P S159E Q206L A216V 1.2

Example 3: Storage Stability Assay II—Stability of Purified Variants

Purification may, for example, be performed as follows:

The culture broth is centrifuged at 26000×g for 20 minutes and thesupernatant is carefully decanted from the precipitate. The supernatantis filtered through a Nalgene 0.2 μm filtration unit in order to removethe remains of the host cells. The pH in the 0.2 μm filtrate is adjustedto pH 8 with 3 M Tris base and the pH-adjusted filtrate is applied to aMEP Hypercel column (Pall Corporation) equilibrated in 20 mM Tris/HCl, 1mM CaCl₂, pH 8.0. After washing the column with the equilibrationbuffer, the column is step-eluted with 20 mM CH₃COOH/NaOH, 1 mM CaCl₂,pH 4.5. Fractions from the column are analyzed for protease activityusing the Suc-AAPF-pNA assay at pH 9 and peak fractions are pooled. ThepH of the pool from the MEP Hypercel column is adjusted to pH 6 with 20%(v/v) CH₃COOH or 3 M Tris base and the pH-adjusted pool is diluted withdeionized water to the same conductivity as 20 mM MES/NaOH, 2 mM CaCl₂,pH 6.0. The diluted pool is applied to an SP-Sepharose® Fast Flow column(GE Healthcare) equilibrated in 20 mM MES/NaOH, 2 mM CaCl₂, pH 6.0.After washing the column with the equilibration buffer, the proteasevariant is eluted with a linear NaCl gradient (0→0.5 M) in the samebuffer over five column volumes. Fractions from the column are analyzedfor protease activity using the Suc-AAPF-pNA assay at pH 9 and activefractions are analyzed by SDS-PAGE. Fractions in which only one band isobserved on the Coomassie stained SDS-PAGE gel are pooled as thepurified preparation and used for further experiments.

Purified protease variants are diluted with 0.01% Triton X-100 to thesame concentration e.g. 0.2 mg/ml, with the concentration calculatede.g. from absorbance at 280 nm. For each protease variant at least 2wells are tested. 30 μl diluted protease sample is mixed with 270 μlconcentrated liquid detergent (e.g. Model A detergent; see thecomposition of this detergent in Table 1 above) in the well of amicrotiter plate (detergent plate, e.g. Nunc U96 PP 0.5 ml) e.g. using amagnetic bar. 20 μl of this mixture is then transferred to anothermicrotiter plate and diluted appropriately with 0.1 M Tris pH 8.6 (where“appropriately” refers to the fact that the dilution should ensure thatthe initial rate of substrate hydrolysis lies within the linear range ofthe assay, which persons skilled in the art will readily be able todetermine). 30 μl of this dilution is transferred to a new microtiterplate, and after addition of 70 μl substrate solution (e.g. 0.72 mg/mlSuc-Ala-Ala-Pro-Phe-pNA (Bachem L-1400) in 0.1 M Tris pH 8.6) theactivity of the unstressed sample is determined from the initial slopeof increase in measured absorbance at 405 nm (e.g. measured every 20 secfor 5 min on a SpectraMax Plus instrument). After sealing, the detergentplate is incubated at a suitable temperature (e.g. 40° C.) in anEppendorf Thermomixer (no shaking). After e.g. 4 and 24 hoursincubation, samples of 20 μl are withdrawn and residual activity ofstressed samples is measured as with the initial unstressed activity.

The decrease in activity during incubation with detergent is assumed tobe exponential. Half-lives (T½) are found from linear regression ofLog(Activity) versus incubation time, and half-life improvement factors(T½ IF) are calculated as the half-life of the protease variant relativeto the half-life of the reference, where the reference is suitably theBPN′ protease having SEQ ID NO: 1.

1. A subtilase variant, comprising mutations at three or more positionsselected from the group consisting of 9, 63, 76, 88, 104, 107, 128, 131,159, 204, 206, 209, 212, 215, 216, 261 and 262, wherein positions arenumbered according to SEQ ID NO: 1, and wherein the variant has proteaseactivity and a sequence identity to SEQ ID NO: 1 of at least 80% andless than 100%.
 2. The subtilase variant of claim 1, wherein the variantcomprises mutations at three or more positions selected from the groupconsisting of: (i) 209 in combination with two or more mutations atpositions selected from 63, 215 and 217; (ii) 104 and 128 in combinationwith at least one mutation at a position selected from 9, 63, 76, 107,131, 159, 204, 206, 212, 215, 216 and 217; (iii) 9 in combination withat least two mutations at positions selected from 76, 204 and 212; (iv)76 in combination with at least two mutations at positions selected from9, 88, 159, 204, 206, 212, 216, 261 and 262; (v) 206 in combination withat least two mutations at positions selected from 9, 76, 159, 204, 209,212, 216, 217, 261 and 262; and (vi) 204, 212 and 216, in combinationwith at least one mutation at a position selected from 9, 159 and 206.3. The subtilase variant of claim 1, wherein: the mutation in position 9is S9E or S9D, the mutation in position 63 is S63G or S63A, the mutationin position 76 is N76D or N76E, the mutation in position 88 is A88V,A88I, A88L or A88M, the mutation in position 104 is Y104V, Y104I, Y104Lor Y104M, the mutation in position 107 is I107L, I107V, I107M, themutation in position 128 is G128S, G128A or G128T, the mutation inposition 131 is G131* or G131P, the mutation in position 159 is S159E orS159D, the mutation in position 204 is S204D or S204E, the mutation inposition 206 is Q206L, Q206I, Q206V or Q206M, the mutation in position209 is L209W, the mutation in position 212 is N212G, N212A or N212S, themutation in position 215 is G215A, the mutation in position 216 isA216V, A216I, A216L or A216M, the mutation in position 217 is Y217L,Y217I, Y217V or Y217M, the mutation in position 261 is F261W, F261N orF261Y, and/or the mutation in position 262 is Y262E or Y262D.
 4. Thesubtilase variant of claim 1, comprising three or more mutationsselected from the group consisting of: L209W in combination with two ormore substitutions selected from S63G/A, G215A and Y217L/I/V/M; (ii)Y104V/I/L/M and G128S/A/T in combination with at least one mutationselected from S9E/D, S63G/A, N76D/E, I107L/V/M, G131*, G131P, S159E/D,S204D/E, Q206L/I/V/M, N212G/A/S, G215A, A216V/I/L/M and Y217L/I/V/M;(iii) S9E/D in combination with at least two substitutions selected fromN76D/E, S204D/E and N212G/A/S; (iv) N76D/E in combination with at leasttwo substitutions selected from S9E/D, A88V/I/L/M, S159E/D, S204D/E,Q206L/I/V/M, N212G/A/S, A216V/I/L/M, F261W/N/Y and Y262E/D; (v)Q206L/I/V/M in combination with at least two substitutions selected fromS9E/D, N76D/E, S159E/D, S204D/E, L209W/N/Y, N212G/A/S, A216V/I/L/M,Y217L/I/V/M, F261W/N/Y and Y262E/D; and (vi) S204D/E, N212G/A/S andA216V/I/L/M, in combination with at least one substitution selected fromS9E/D, S159E/D and Q206L/I/V/M.
 5. A subtilase variant, comprising thesubstitution L209W and at least two substitutions selected from thegroup consisting of S63G/A, G215A and Y217L/I/V/M, wherein positions arenumbered according to SEQ ID NO: 1, and wherein the variant has proteaseactivity and a sequence identity to SEQ ID NO: 1 of at least 80% andless than 100%.
 6. The subtilase variant of claim 5, comprising thesubstitutions S63G, L209W, G215A and Y217L.
 7. The subtilase variant ofclaim 5, further comprising at least one mutation selected from thegroup consisting of S9E/D, N76D/E, Y104V/I/L/M, G128S/A/T, G131*, G131P,S204D/E, Q206L/I/V/M, A216V/I/L/M, F261W/N/Y and Y262E/D.
 8. Thesubtilase variant of claim 5, comprising one of the following sets ofmutations: S63G L209W G215A Y217L; S9E S63G L209W G215A Y217L; S63G N76DL209W G215A Y217L; S63G S204D L209W G215A Y217L; S63G Q206L L209W G215AA216V Y217L; S63G Q206L L209W G215A Y217L; S63G L209W G215A Y217L F261WY262E; S63G Y104V G128S G131* L209W G215A Y217L; or S63G Y104V G128SG131P L209W G215A Y217L.
 9. A subtilase variant, comprising thesubstitutions Y104V/I/L/M+G128S/A/T, and (a) at least two mutationsselected from the group consisting of S9E/D, N76D/E, I107L/V/M, G131*,G131P, S159E/D, S204D/E, Q206L/I/V/M, N212G/A/S and A216V/I/L/M, or (b)L209W and at least two substitutions selected from the group consistingof S63G/A, G215A and Y217L/I/V/M, wherein positions are numberedaccording to SEQ ID NO: 1, and wherein the variant has protease activityand a sequence identity to SEQ ID NO: 1 of at least 80% and less than100%.
 10. The subtilase variant of claim 9, comprising one of thefollowing sets of mutations: N76D Y104V G128S G131* N212G; N76D Y104VG128S G131P; N76D Y104V G128S G131*; Y104V I107L G128S G131* Q206L; N76DY104V G128S G131P N212G; S63G Y104V G128S G131* L209W G215A Y217L; Y104VG128S G131* Q206L A216V; S63G Y104V G128S G131P L209W G215A Y217L; Y104VG128S G131* Q206L; Y104V G128S G131* S159E Q206L A216V; Y104V G128SG131* S204D N212G; Y104V G128S G131P S204D N212G; Y104V G128S G131PQ206L A216V; S9E Y104V G128S G131*; Y104V G128S G131P Q206L; Y104V G128SG131P S159E Q206L; S9E Y104V G128S G131P; or Y104V G128S G131P S159EQ206L A216V.
 11. A subtilase variant, comprising at least threemutations selected from the group consisting of S9E/D, N76D/E,A88V/I/L/M, S159E/D, S204D/E, Q206L/I/V/M, L209W, N212G/A/S,A216V/I/L/M, Y217L/I/V/M, F261W/N/T and Y262E/D, wherein positions arenumbered according to SEQ ID NO: 1, and wherein the variant has proteaseactivity and a sequence identity to SEQ ID NO: 1 of at least 80% andless than 100%.
 12. The subtilase variant of claim 11, comprising one ofthe following sets of mutations: N76D S159E Q206L N212G A216V; S204DQ206L N212G A216V; Q206L N212G A216V; S159E Q206L N212G A216V; S9E S159EQ206L A216V; N76D N212G F261W Y262E; S204D Q206L N212G; S9E S204D N212G;N76D A88V S204D N212G; Q206L F261W Y262E; or Q206L L209W A216V Y217L.13. The subtilase variant of claim 1, wherein the variant has anincreased storage stability in a detergent composition compared to thesubtilase of SEQ ID NO:
 1. 14. The subtilase variant of claim 13,wherein the variant has an increased storage stability in a detergentcomposition compared to the subtilase of SEQ ID NO: 1 when measured for24 hours as described in storage stability assay I in Example 2 herein,and/or as measured as described in storage stability assay II in Example3 herein.
 15. The subtilase variant of claim 14, wherein the variant hasan increased storage stability in a detergent composition compared tothe subtilase of SEQ ID NO: 1 of at least 25% when measured for 24 hoursas described in storage stability assay I in Example 3 herein, and/or asmeasured as described in storage stability assay II in Example 4 herein.16. A method for stabilizing a subtilase variant, the method comprisingintroducing into a parent subtilase having protease activity and atleast 80% sequence identity to SEQ ID NO: 1 at least three mutationsselected from: L209W in combination with two or more substitutionsselected from S63G/A, G215A and Y217L/I/V/M; (ii) Y104V/I/L/M andG128S/A/T in combination with at least one mutation selected from S9E/D,S63G/A, N76D/E, I107L/V/M, G131*, G131P, S159E/D, S204D/E, Q206L/I/V/M,N212G/A/S, G215A, A216V/I/L/M and Y217L/I/V/M; (iii) S9E/D incombination with at least two substitutions selected from N76D/E,S204D/E and N212G/A/S; (iv) N76D/E in combination with at least twosubstitutions selected from S9E/D, A88V/I/L/M, S159E/D, S204D/E,Q206L/I/V/M, N212G/A/S, A216V/I/L/M, F261W/N/Y and Y262E/D; (v)Q206L/I/V/M in combination with at least two substitutions selected fromS9E/D, N76D/E, S159E/D, S204D/E, L209W/N/Y, N212G/A/S, A216V/I/L/M,Y217L/l/V/M, F261W/N/Y and Y262E/D; and (vi) S204D/E, N212G/A/S andA216V/I/L/M, preferably in combination with at least one substitutionselected from S9E/D, S159E/D and Q206L/I/V/M.
 17. The method of claim16, wherein the stabilized subtilase variant has an increased storagestability in a detergent composition compared to the subtilase of SEQ IDNO:
 1. 18. The method of claim 17, wherein the variant has an increasedstorage stability in a detergent composition compared to the subtilaseof SEQ ID NO: 1 when measured for 24 hours as described in storagestability assay I in Example 3 herein, and/or as measured as describedin storage stability assay II in Example 4 herein.
 19. The method ofclaim 18, wherein the variant has an increased storage stability in adetergent composition compared to the subtilase of SEQ ID NO: 1 of atleast 25% when measured for 24 hours as described in storage stabilityassay I in Example 3 herein, and/or as measured as described in storagestability assay II in Example 4 herein.
 20. A method for obtaining asubtilase variant, the method comprising (a) providing a host cellcomprising a polynucleotide encoding a variant of a parent proteasehaving three or more mutations, e.g. four or more mutations, compared toSEQ ID NO: 1, wherein the variant has protease activity and a sequenceidentity to SEQ ID NO: 1 of at least 80%, and wherein the mutations areselected from: L209W in combination with two or more substitutionsselected from S63G/A, G215A and Y217L/I/V/M; (ii) Y104V/I/L/M andG128S/A/T in combination with at least one mutation selected from S9E/D,S63G/A, N76D/E, I107L/V/M, G131*, G131P, S159E/D, S204D/E, Q206L/I/V/M,N212G/A/S, G215A, A216V/I/L/M and Y217L/I/V/M; (iii) S9E/D incombination with at least two substitutions selected from N76D/E,S204D/E and N212G/A/S; (iv) N76D/E in combination with at least twosubstitutions selected from S9E/D, A88V/I/L/M, S159E/D, S204D/E,Q206L/I/V/M, N212G/A/S, A216V/I/L/M, F261W/N/Y and Y262E/D; (v)Q206L/I/V/M in combination with at least two substitutions selected fromS9E/D, N76D/E, S159E/D, S204D/E, L209W/N/Y, N212G/A/S, A216V/I/L/M,Y217L/I/V/M, F261W/N/Y and Y262E/D; and (vi) S204D/E, N212G/A/S andA216V/I/L/M, preferably in combination with at least one substitutionselected from S9E/D, S159E/D and Q206L/I/V/M; (b) cultivating the hostcell under conditions suitable for expression of the variant; and (c)recovering the variant.
 21. A detergent composition comprising asubtilase variant according to claim 1 and at least one detergentcomponent, and optionally further comprising at least one additionalenzyme.
 22. (canceled)